US 8133859 B2
The present disclosure is generally related to methods of using the secretory protein SCGB3A2 for promoting lung development and treating lung disease. Some embodiments are, for example, methods for treating and inhibiting the development of neonatal respiratory distress. Other embodiments are methods of promoting lung development in damaged or diseased lungs. Also disclosed are methods for inhibiting lung damage due to anti-cancer agents.
1. A method of promoting lung development in a fetus, comprising:
administering a therapeutically effective amount of murine or human SCGB3A2 to a pregnant mouse or human subject, wherein administration of the SCGB3A2 to the subject results in administration of the SCGB3A2 to the fetus in the pregnant subject, and wherein the administration of the SCGB3A2 to the fetus increases lung branching morphogenesis in the fetus, thereby promoting lung development in the fetus.
2. The method of
3. The method of
4. The method of
5. A method of promoting lung development ex vivo, comprising, culturing a murine or human lung explant with an effective amount of murine or human SCGB3A2, wherein culturing the lung explant increases lung branching morphogenesis in the lung explant, thereby promoting lung development ex vivo.
6. The method of
7. The method of
This is the U.S. National Stage of International Application No. PCT/US2007/079771, filed Sep. 27, 2007, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Patent Application No. 60/847,747, filed Sep. 27, 2006, and U.S. Provisional Patent Application No. 60/880,134, filed Jan. 12, 2007. Each of these applications is incorporated by reference herein in its entirety.
The present disclosure is generally related to methods of promoting lung development and treating lung disease, for example, methods for treating and inhibiting the development of neonatal respiratory distress, methods of promoting lung development, and methods for inhibiting lung damage due to anti-cancer agents.
Neonatal respiratory distress syndrome (RDS), also called respiratory distress syndrome of prematurity, is a syndrome caused by developmental lack of surfactant and structural immaturity in the lungs of premature infants. RDS affects about 1% of newborn infants. The incidence decreases with advancing gestational age, from about 50% in babies born at 26-28 weeks, to about 25% at 30-31 weeks. The syndrome is more frequent in infants of diabetic mothers and in the second born of premature twins. Despite huge advances in care, RDS remains the most common single cause of death in the first month of life. Complications include metabolic disorders (acidosis, low blood sugar), patent ductus arteriosus, low blood pressure, chronic lung changes, and intracranial hemorrhage.
Pulmonary fibrosis is a disease of inflammation that results in scarring, or fibrosis, of the lungs. In time, this fibrosis can build up to the point where the lungs are unable to provide oxygen to the tissues of the body. The average survival rate for a subject with pulmonary fibrosis is about four to six years after diagnosis. Pulmonary fibrosis is the most common side effect of certain anti-cancer agents, such as cytotoxic antibiotics.
Given the foregoing, it would be desirable to have effective methods for treating or preventing neonatal respiratory distress syndrome, and methods of inhibiting the development of pulmonary fibrosis resulting from exposure to anti-cancer agents such as cytotoxic antibiotics.
Disclosed herein are methods for treating and inhibiting the development of neonatal respiratory distress, methods of promoting lung development, and methods of inhibiting or reducing lung damage that results from treatment with certain anti-cancer agents. These methods are based on the surprising discovery that the secretory protein SCGB3A2 has both growth factor and anti-apoptotic activities, in addition to its previously-known anti-inflammatory activities. Examples of these methods are provided in the context of a specific example of a SCGB3A2 protein; however, other equivalents are contemplated.
One such method of treatment is a method for using SCGB3A2 to treat or inhibit the development of neonatal respiratory distress in a subject. In some embodiments, the SCGB3A2 is administered prenatally, and in other embodiments the SCGB3A2 is administered postnatally.
Also disclosed herein is a method of promoting lung development. The method includes contacting a lung cell with an effective amount of SCGB3A2. In various embodiments the method is carried out in vitro, whereas in other embodiments the method is carried out in vivo.
Yet another method is disclosed for using SCGB3A2 to inhibit or reduce lung damage in a subject being treated with an anti-cancer agent. In some embodiments, the anticancer agent is a cytotoxic antibiotic, and in particular examples, the anti-cancer agent is bleomycin.
The foregoing and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.
SEQ ID NO: 1 is a sense Rad23b siRNA probe 1.
SEQ ID NO: 2 is an antisense Rad23b siRNA probe 1.
SEQ ID NO: 3 is a sense Rad23b siRNA probe 2.
SEQ ID NO: 4 is an antisense Rad23b siRNA probe 2.
SEQ ID NO: 5 is a negative sense siRNA.
SEQ ID NO: 6 is a negative sense siRNA.
SEQ ID NO: 7 is a Rad23b primer (forward).
SEQ ID NO: 8 is a Rad23b primer (reverse).
SEQ ID NO: 9 is an FGF2 primer (forward).
SEQ ID NO: 10 is an FGF2 primer (reverse.
SEQ ID NO: 11 is an FGF7 primer (forward).
SEQ ID NO: 12 is an FGF7 primer (reverse).
SEQ ID NO: 13 is an FGF9 primer (forward).
SEQ ID NO: 14 is an FGF9 primer (reverse).
SEQ ID NO: 15 is an FGF10 primer (forward).
SEQ ID NO: 16 is an FGF10 primer (reverse).
SEQ ID NO: 17 is an 18S primer (forward).
SEQ ID NO: 18 is an 18S primer (reverse).
SEQ ID NO: 19 is oligonucleotide L2575.
SEQ ID NO: 20 is oligonucleotide L2576.
SEQ ID NO: 21 is oligonucleotide L907.
SEQ ID NO: 22 is a linker amino acid sequence.
SEQ ID NO: 23 is an SP-A primer (forward).
SEQ ID NO: 24 is an SP-A primer (reverse).
SEQ ID NO: 25 is an SP-D primer (forward).
SEQ ID NO: 26 is an SP-D primer (reverse).
SEQ ID NO: 27 is an AQP1 primer (forward).
SEQ ID NO: 28 is an AQP1 primer (reverse).
SEQ ID NO: 29 is an OB-R primer (forward).
SEQ ID NO: 30 is an OB-R primer (reverse).
Disclosed herein are therapeutic methods that make use of the secretory protein SCGB3A2 and equivalents and analogs thereof. These methods are based on the surprising discovery that SCGB3A2 has both growth factor and anti-apoptotic activities, in addition to its previously-known anti-inflammatory activities.
Some embodiments are methods of treating or preventing neonatal respiratory distress in a subject. These methods include administering to the subject an effective amount of SCGB3A2, thereby treating or preventing the neonatal respiratory distress. In certain examples of these methods, the SCGB3A2 is administered prenatally, for instance transplacentally or amniotically. In other embodiments, the SCGB3A2 is administered postnatally, for instance intravenously, intra-arterially, intra-peritoneally, subcutaneously, or by inhalation.
Other methods are methods of promoting lung development. These methods include contacting a lung cell with an effective amount of SCGB3A2, thereby promoting lung development. In some examples of the methods, contacting the lung cell with an effective amount of SCGB3A2 occurs ex vivo, and in particular examples the lung cell is in a component of an explant. Still more particular examples include a further step of transplanting the lung cell (or explant, for instance) into a subject after the cell is contacted with SCBG3A2. In other embodiments of the methods, contacting the lung cell with an effective amount of SCGB3A2 occurs via administering the SCGB3A2 to a subject, for example transplacentally, amniotically, intravenously, intra-arterially, intra-peritoneally, subcutaneously, or by inhalation. In certain examples, the subject has diminished lung capacity, and in more particular examples the subject is a neonate.
Also disclosed are methods of inhibiting or reducing lung damage in a subject being treated with an anti-cancer agent. These methods include administering to the subject an effective amount of SCGB3A2, thereby inhibiting lung damage caused by the anti-cancer agent. In certain embodiments of the method, the SCGB3A2 is administered intravenously, intra-arterially, intra-peritoneally, subcutaneously, or by inhalation, and in particular examples, the SCGB3A2 is administered in conjunction with the anti-cancer agent. In even more particular examples, the anti-cancer agent is a cytotoxic antibiotic, and in some instances, the anti-cancer agent is bleomycin. In certain examples, the lung damage includes pulmonary fibrosis.
The methods disclosed herein are provided in the context of a specific example SCGB3A2 protein, however, other equivalents are contemplated. For instance, animal equivalents of SCGB3A2 would be appropriate for use in veterinary situations, or in animal models of lung development, pulmonary fibrosis, and neonatal respiratory distress, and these and other equivalents also may be used in human therapies.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
In order to facilitate review of the various embodiments of the disclosure, the following explanation of terms is provided:
Acute respiratory distress syndrome (ARDS): An inflammatory lung disease characterized by acute hypoxemia respiratory failure due to pulmonary edema (reviewed in Honing & Ingram, in: Harrison's Principles of Internal Medicine, 14th Edition, A. S. Fauci, et al. (eds.), McGraw-Hill, N.Y., pp. 1483-1486, 1998; and Goodman et al., Am J. Respir. Crit. Care Med. 154:602-11, 1996). ARDS represents a spectrum of responses to acute lung injury (ALI); these responses occur as complications of a more widespread systemic response to acute inflammation or injury.
ALI develops rapidly after a predisposing condition triggers a systemic inflammatory response, and is most strongly associated with conditions that produce direct alveolar injury or direct injury via the pulmonary capillary bed, such as aspiration, diffuse infection, toxic inhalation, direct injury to the alveolar epithelium, or sepsis syndrome. ALI is the consequence of unregulated over-expression of usual systemic inflammatory responses to infection and/or injury. Injury involves the alveolar epithelium and the pulmonary capillary endothelium, and results in a complex cascade of events. Injury is produced by cellular events associated with neutrophils, macrophages, monocytes, and lymphocytes producing various cytokines, in turn producing cellular activation, chemotaxis, and adhesion.
ARDS can be distinguished from neonatal respiratory distress syndrome, which occurs in newborn infants, and which is a consequence of lung immaturity and lack of sufficient lung surfactant.
Amniotic administration: Administration (to the fetus) via the amniotic fluid. In amniotic administration, an agent is administered maternally, and is absorbed by the fetus from the amniotic fluid. In some examples, an agent is absorbed through fetal skin, and in other examples, it is absorbed through the lungs. Administration to the mother of a compound that is to be delivered to a fetus via amniotic administration includes both oral and parenteral routes of administration, and can include direct injection or infusion into the amniotic sac.
Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects. Therefore, the general term “subject” is understood to include all animals, including, but not limited to, humans, or veterinary subjects, such as other primates, dogs, cats, horses, and cows.
Anti-cancer agent: An anti-neoplastic agent. Anti-cancer agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and bbr3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example, capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and other agents, such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, pentostatin, masoprocol, mitotane, pegaspargase, and tretinoin.
Apoptosis: A process of cellular suicide. Apoptosis is one of the main types of programmed cell death, and involves an orchestrated series of biochemical events leading to a characteristic cell morphology and death. The apoptotic process is executed in such a way as to safely dispose of cellular debris.
In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis is carried out in an orderly process that generally confers advantages during an organism's life cycle. For example, the differentiation of fingers and toes in a developing human embryo requires cells between the fingers to initiate apoptosis so that the digits can separate. Between 50 billion and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 to 14, approximately 20 billion to 30 billion cells die a day.
Defective apoptotic processes have been implicated in an extensive variety of diseases. Excessive apoptosis causes hypotrophy, such as in ischemic damage, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.
Bleomycin: A glycosylated linear nonribosomal peptide antibiotic produced by the bacterium Streptomyces verticillus. More generally, the term bleomycin refers to a family of structurally related compounds. When used as an anti-cancer agent, the chemotherapeutical forms are primarily bleomycin A2 and B2. The drug is used in the treatment of Hodgkin's lymphoma (as a component of the adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) regimen), squamous cell carcinomas, and testicular cancer as well as pleurodesis, for instance.
A serious complication of bleomycin treatment is pulmonary fibrosis, which is an inflammatory disease that results in scarring, or fibrosis, of the lungs. In time, this fibrosis can build up to the point where the lungs are unable to provide sufficient oxygen to the tissues of the body. Pulmonary fibrosis sets off a series of events in which the inflammation and immune activity in the lungs, and eventually the fibrosis processes, become uncontrollable.
Bronchodilator: An antispasmodic or other agent that dilates a bronchus or bronchiole. Bronchodilators relax the smooth muscles of the airways, allowing the airway to dilate. Bronchodilator medicines do not counteract inflammation.
Chronic Bronchitis: An inflammatory lung disease that results in a long-standing inflammation of the airways that produces a lot of mucus, causing wheezing and infections. It is considered chronic if a subject has coughing and mucus on a regular basis for at least three months a year and for two years in a row.
Chronic Obstructive Pulmonary Disease (COPD): A lung disease that encompasses two closely related respiratory disorders that cause gradual loss of pulmonary function: chronic bronchitis and emphysema. A subject with COPD sometimes has both chronic bronchitis and emphysema, or may just have one of these diseases. Emphysema is an inflammatory lung disease that destroys the alveolae and/or bronchae. Over time, the lungs lose elasticity. This causes the air sacs to become enlarged, thus making breathing difficult.
In the beginning stages of COPD, a subject may have only a mild shortness of breath and occasional coughing spells. Initial symptoms can include a general feeling of illness, increasing shortness of breath, coughing, and wheezing. But, as the disease progresses, symptoms become increasingly more severe.
The overwhelming cause of COPD is smoking. Approximately 90% of COPD subjects have a history of smoking. In addition, untreated or under-treated asthma may lead to irreversible lung damage. These subjects may have symptoms similar to COPD.
Cystic Fibrosis: An inflammatory lung disease that also affects the lungs and digestive systems, especially the pancreas. It causes the exocrine glands, which produce mucus and sweat, to produce abnormal secretions. Cystic fibrosis causes the cells in the lung tissue to produce an abnormal amount of thick, sticky mucus that clogs the airways of the lungs, resulting in pulmonary obstructions and life-threatening bacterial infections.
Cytotoxic antibiotic: A class of anti-cancer drugs that includes anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, hydroxyurea, and mitomycin. The most serious complication of treatment with cytotoxic antibiotics is pulmonary fibrosis, which is an inflammatory disease that results in scarring, or fibrosis, of the lungs. In time, this fibrosis can build up to the point where the lungs are unable to provide oxygen to the tissues of the body. Pulmonary fibrosis sets off a series of events in which the inflammation and immune activity in the lungs, and eventually the fibrosis processes, become uncontrollable.
DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Thus, a reference to the nucleic acid molecule that encodes SCGB3A2 (for instance. UGRP1 type A mRNA: AF274959, type B mRNA: AF274960, type C mRNA: AF274961, mUGRP2: AF313456, EST AI391046, hUGRP1: AF313455, EST AI355612, EST AI355302, and hUGRP2: AF313458, EST AW974727), or a fragment thereof, encompasses both the sense strand and its reverse complement. Thus, for instance, it is appropriate to generate probes or primers from the reverse complement sequence of the disclosed nucleic acid molecules.
Expectorant: A drug or chemical substance that induces the ejection of mucus, phlegm, and other fluids from the lungs and air passages, for example by coughing.
Expiratory Flow Rate: The rate at which air is expelled from the lungs during exhalation. A subject's maximum expiratory flow is measured by a simple pulmonary test; in performing the test, a subject first takes as deep a breath as possible, then exhales as rapidly and as completely as possible into a machine known as a spirometer, which measures the rate of exhalation. Forced expiratory flow 25-75 (FEF 25-75) is a measurement of the forced expiratory flow determined over the midportion of a forced exhalation. An increase in the forced expiratory flow (FEF) or FEF 25-75 reflects a decrease in bronchoconstriction and an improvement in pulmonary function.
Forced Expiratory Volume (FEV; FEV1): The volume of air resulting from the forced expiratory flow test, in which a subject first inspires maximally to the total lung capacity, then exhales as rapidly and as completely as possible. The forced expired volume in one second (FEV1) represents the maximum expiratory air volume a subject can produce during a one-second interval. An increase in FEV or FEV1 reflects a decrease in bronchoconstriction and an improvement in pulmonary function.
Forced Vital Capacity (FVC): The volume of air resulting from the forced expiratory flow test, in which a subject first inspires maximally to the total lung capacity, then exhales as rapidly and as completely as possible. An increase in FVC reflects a decrease in bronchoconstriction and an improvement in pulmonary function.
Growth factor: A protein capable of stimulating cellular proliferation, differentiation, and/or commitment. The term growth factor is sometimes used interchangeably with the term cytokine. Historically, cytokines were associated with hematopoietic (blood forming) cells and immune system cells (for instance, lymphocytes and tissue cells from spleen, thymus, and lymph nodes). However, some of the same signaling proteins used by the hematopoietic and immune systems are used by other cells and tissues, both during development and in the mature organism.
Some specific, non-limiting examples of growth factors include Transforming growth factor beta (TGF-β). Granulocyte-colony stimulating factor (G-CSF), Granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), Neurotrophins (for instance, NGF, BDNF, and NT3), Platelet-derived growth factor (PDGF), Erythropoietin (EPO), Thrombopoietin (TPO), Myostatin (GDF-8), Growth differentiation factor-9 (GDF9), Basic fibroblast growth factor (bFGF or FGF2), Epidermal growth factor (EGF), Hepatocyte growth factor (HGF), and, as described herein, SCGB3A2.
Glucocorticoid: A class of steroid hormones characterized by an ability to bind with the cortisol receptor and trigger similar effects. Glucocorticoids have potent anti-inflammatory and immunosuppressive properties. As a consequence, glucocorticoids are widely used as drugs to treat inflammatory conditions such as inflammatory lung diseases. In addition, glucocorticoids have multiple effects on fetal development, including promoting maturation of the lung and production of the surfactant necessary for extrauterine lung function. Thus, they are often administered to women in pre-term labor in order to attempt to speed lung maturation in a pre-term infant.
Specific, non-limiting examples of glucocorticoids include hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate, and aldosterone.
Inflammatory lung disease: A disease of the lung that is associated with lung inflammation. In many inflammatory lung diseases, the inflammatory response that accompanies the underlying disease state is much more dangerous than the underlying infection or trauma. Inflammatory lung diseases can include, but are not limited to pneumonia, ARDS, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pulmonary fibrosis, and pulmonary sarcoidosis. For the purposes of this disclosure, the term inflammatory lung disease does not include neonatal respiratory distress syndrome, which is a condition involving lung immaturity and lack of surfactants.
Inspiratory Flow Rate: The rate at which air travels into the lungs during inspiration. Inspiratory flow is measured by a simple pulmonary test; in performing the test the subject takes as deep and rapid a breath as possible from a machine known as a spirometer, which measures the rate of inspiration. An increase in inspiratory flow rate reflects a decrease in bronchoconstriction and an improvement in pulmonary function.
Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
Lung damage: Damage or insult to the lungs or respiratory system. Lung damage includes direct airway or alveolar injury or direct injury via the pulmonary capillary bed, such as aspiration, diffuse infection, toxic inhalation, direct injury to the alveolar epithelium, or sepsis syndrome. Lung damage also includes lung-related drug effects, such as the pulmonary fibrosis that can result from exposure to certain anti-cancer agents, such as the cytotoxic antibiotic bleomycin.
Lung Volume: The maximum volume the lungs can hold.
Nanoparticles: Solid colloidial particles that range in size from about 10-1000 nm. They can be made from biodegradable and biocompatible biomaterials. Active components, such as drugs, can be adsorbed, encapsulated, or covalently attached to their surface or into their matrix.
Neonate: A human or animal infant less than about a month old. The term “neonate” includes premature infants and postmature infants, as well as full term newborns.
Neonatal Respiratory Distress: Also called respiratory distress of prematurity and respiratory distress syndrome (RDS), neonatal respiratory distress results from a lack of pulmonary surfactant, a molecular substance that helps the lung's alveoli (air sacs) do their job of extracting carbon dioxide from the blood and replacing it with oxygen. The surfactant prevents the lung's alveoli from collapsing and helps keep them properly inflated by reducing their surface tension. The absence of surfactant prevents the alveoli from functioning properly. RDS affects about 1% of newborn infants. The incidence decreases with advancing gestational age (length of pregnancy), from about 50% in babies born at 26-28 weeks, to about 25% at 30-31 weeks. The syndrome is more frequent in infants of diabetic mothers and in the second born of premature twins.
Respiratory distress syndrome begins shortly after birth, and is manifested by tachypnea and “sucking in” (retractions) of the chest wall during breathing efforts (respiratory distress). In addition grunting respirations, flaring of the nostrils and cyanosis (a blue discoloration of the skin due to low oxygen content in the blood) are frequent. As the disease progresses, the neonate may develop ventilatory failure (climbing carbon dioxide concentrations in the blood), and prolonged cessations of breathing (“apnea”). Whether treated or not, the clinical course for the acute disease lasts about two to three days. During the first, the patient worsens and requires more support. During the second the subject may be remarkably stable on adequate support and resolution is noted during the third day, heralded by a prompt diuresis. Despite huge advances in care, RDS remains the most common single cause of death in the first month of life. Complications include metabolic disorders (acidosis, low blood sugar), patent ductus arteriosus, low blood pressure, chronic lung changes, and intracranial hemorrhage.
The lungs of an infant with RDS are developmentally deficient in a material called surfactant, which allows the alveoli to remain open throughout the normal cycle of inhalation and exhalation. Surfactant is a complex system of lipids, proteins and glycoproteins which are produced in specialized lung cells called Type II cells or Type II pneumocytes. The surfactant is packaged by the cell in structures called lamellar bodies, and extruded into the alveoli. The lamellar bodies then unfold into a complex lining of the alveoli. This layer serves the purpose of reducing the surface tension which would tend to cause the alveoli to collapse in the presence of gas. Without adequate amounts of surfactant, the alveoli collapse and are very difficult to expand.
Structural immaturity, as manifested by low numbers of alveoli, also contributes to the disease process. Additionally, the oxygen and breathing treatments used, while life-saving, can also damage the lungs. The diagnosis of RDS is made by the clinical picture and the chest x-ray, which demonstrates decreased lung volumes (bell-shaped chest), absence of the thymus (after about six hours), a small (0.5-1 mm), discrete, uniform infiltrate involving all lobes of the lung and air-bronchograms. In severe cases, this becomes exaggerated until the cardiac borders become inapparent (‘white-out’ or ‘ground-glass’).
Parenteral: Administered outside of the intestine, e.g., not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered amniotically, transplacentally, intravenously, intrathecally, intramuscularly, intraperitoneally, intra-articularly, or subcutaneously, and various surface applications including intranasal, inhalational, intradermal, and topical application, for instance.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Martin, Remington's Pharmaceutical Sciences, published by Mack Publishing Co. Easton, Pa., 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of, for instance SCGB3A2.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Pneumonia: An inflammatory lung disease characterized by inflammation and consolidation followed by resolution and caused by infection from viruses, fungi, or physical and chemical irritants or bacteria including: Pneumonococcus, Streptococcus, Hemolyticus, Staphylococcus, Friedländer's bacillus, and influenza bacillus. Symptoms include high fever, chest pain, difficulty breathing, coughing and sputum.
Preventing or treating a disease: “Preventing” a disease refers to inhibiting the full development of a disease, for example in a person who is at risk for a disease such as neonatal respiratory distress or bleomycin-induced pulmonary fibrosis. An example of a subject at risk for neonatal respiratory distress is a neonate born at less than forty weeks' gestation, a fetus at risk of premature birth, or a fetus or neonate born to a mother with a condition that may cause delayed lung development, such as diabetes. An example of a subject at risk for developing pulmonary fibrosis is someone who has begun or will begin treatment with an anti-cancer agent that can cause pulmonary fibrosis, such as bleomycin. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
Pulmonary Fibrosis: An inflammatory lung disease that results in scarring, or fibrosis, of the lungs. In time, this fibrosis can build up to the point where the lungs are unable to provide oxygen to the tissues of the body. Pulmonary fibrosis can result from an autoimmune disorder, the after effects of an infection, such as a viral infection, or from exposure to certain anti-cancer agents, such as cytotoxic antibiotics. A specific example of an anti-cancer agent that can cause pulmonary fibrosis is bleomycin.
Pulmonary fibrosis sets off a series of events in which the inflammation and immune activity in the lungs, and eventually the fibrosis processes, become uncontrollable. The average survival rate for a subject with pulmonary fibrosis is about four to six years after diagnosis. Those who develop pulmonary fibrosis at a young age seem to have a longer survival than those who develop it later in life.
Pulmonary function: The function of the respiratory system, which can be measured through a variety of tests, including, but not limited to measurements of airflow (e.g. spirometry) or arterial blood gases. Measurements of airflow included airflow rate, peak expiratory flow rate (PEFR), forced expiratory volume in the first second (FEV1), and maximal midexpiratory rate (MMEFR).
Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate).
Rad23b: One of the two Mus musculus homologs of the Saccharomyces cerevisiae DNA repair protein RAD23. Rad23b is tightly complexed with xeroderma pigmentosum group C(XPC), serving as a primary DNA damage sensor.
Respiratory Disorder: A large variety of abnormalities arising in all structures of the body involved with gas exchange. These structures include the lungs, nose, oropharynx, extrapulmonary airways, thoracic cage, and respiratory muscles. Respiratory disorders encompass both acute and chronic diseases. Asthma is one specific, non-limiting example of a respiratory disorder. Other specific, non-limiting examples include coughs, pneumonia, bronchitis (for example, chronic obstructive bronchitis), neonatal respiratory distress, pulmonary fibrosis, emphysema, interstitial lung disease, cystic fibrosis, and lung tumors.
SCGB3A2: Also called uteroglobin-related protein 1 (UGRP1), LuLeu1, lu103, Pnsp1, and Hin-2, SCGB3A2 is a member of the uteroglobin/Clara cell secretory protein (UG/CCSP) gene superfamily of secretory proteins, and is predominantly expressed in the epithelial cells of trachea, bronchus, and bronchioles. SCGB3A2 has been shown to suppress lung inflammation using a mouse model for allergic airway inflammation (Chiba et al., (2006) Am. J. Respir. Crit. Care Med May 1; 173(9):958-64.). MARCO, a macrophage scavenger receptor that is expressed in lung alveolar macrophages and is involved in pulmonary inflammation, has been identified as the receptor for SCGB3A2 (Bin et al., (2003) J. Immunol. 171, 924-30).
As used herein, the term “SCGB3A2” is intended generally. Thus, in one embodiment, SCGB3A2 is a human protein. In another embodiment, SCGB3A2 is a non-human animal homolog/ortholog of the human molecule, such as a sheep, chimpanzee, goat, pig, mouse, rat, or hamster SCGB3A2-equivalent protein.
The human SCGB3A2 gene is about 2,900 base pairs in length and consists of three exons. The first intron of SCGB3A2 is about five to six-fold longer than the second intron, which resembles the structure of orthologous mouse SCGB3A2 gene. Specific, non-limiting examples of SCGB3A2 nucleotide sequences include the following GenBank accession numbers; UGRP1 type A mRNA: AF274959, type B mRNA: AF274960, type C mRNA: AF274961, mUGRP2: AF313456, EST AI391046, hUGRP1: AF313455, EST AI355612, EST AI355302, and hUGRP2: AF313458, EST AW974727. One of ordinary skill in the art will recognize that these are provided merely as examples; other proteins/nucleic acids that fall into the described class will be recognized.
Stem Cells: Primal cells found in all multi-cellular organisms. Stem cells retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types. The three broad categories of mammalian stem cells are: embryonic stem cells, which are derived from blastocysts; adult stem cells, which are found in adult tissues; and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells.
By definition, stem cells possess two properties: self-renewal, the ability to go through numerous cycles of cell division while maintaining the undifferentiated state; and unlimited potency, the capacity to differentiate into any mature cell type. In a strict sense, this requires stem cells to be either totipotent or pluripotent, although some multipotent and/or unipotent progenitor cells are sometimes referred to as stem cells.
Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell. Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. These stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers.
Multipotent stem cells can produce only cells of a closely related family of cells (for instance, hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self-renewal, which distinguishes them from non-stem cells.
The term progenitor cell refers to immature or undifferentiated cells, typically found in postnatal animals. While progenitor cells share many common features with stem cells, the term is far less restrictive. Like stem cells, progenitor cells have a capacity for self-renewal and differentiation, although these properties may be limited. The majority of progenitor cells lie dormant or possess little activity in the tissue in which they reside. They exhibit slow growth and their main role is to replace cells lost by normal attrition. Upon tissue damage or injury, progenitor cells can be activated by growth factors or cytokines, leading to increased cell division important for the repair process.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals. The methods disclosed herein have equal applications in medical and veterinary settings. Therefore, the general term “subject” is understood to include all animals, including, but not limited to, humans or veterinary subjects, such as other primates, dogs, cats, horses, and cows.
Therapeutically effective amount: A quantity of a specified compound (such as SCGB3A2 or an equivalent thereof) required to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to treat or prevent neonatal respiratory distress or bleomycin-induced pulmonary fibrosis in a subject, or a dose sufficient to prevent advancement, or to cause regression of a disease, or which is capable of relieving symptoms caused by a disease, such as pain, lung inflammation, fluid accumulation, or shortness of breath.
Transplacental administration: Administration of an agent to a fetus via the placenta. The agent can be administered to the mother via any route of administration, including topical, oral, and parenteral routes of administration. An agent that is administered by transplacental administration crosses from the maternal bloodstream, across the placenta, and into the fetal bloodstream.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a”, “an”, and “the” include plural referents unless context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Lung tissue arises by budding from the ventral foregut at approximately embryonic day (E) 9.5 in mouse gestation (Kaufman & Bard (1999) The Anatomical Basis of Mouse Development, Academic Press, London). Mouse lung development is classified as four stages; pseudoglandular (E9.5-16.5), canalicular (E16.5-17.5), terminal saccular (E17.5-perinatal day (P) 5), and alveolar (P5-30) (Perl et al., (1999) Clin. Genet. 56, 14-27; Warburton et al., (2000) Mech. Dev. 92, 55-81). This classification is representative of the complexity of morphological and functional changes that occur during lung development.
Lung development is controlled temporally and spatially by various transcription factors and growth factors (Perl et al., (1999) Clin. Genet. 56, 14-27; Cardoso (2001) Annu. Rev. Physiol. 63, 471-94). Among them, TITF1, also called T/EBP, TTF1 or NKX2.1, is expressed in lung, thyroid and ventral forebrain during early embryogenesis (Lazzaro et al., (1991) Development 113, 1093-104; Kimura et al., (1996) Genes Dev. 10, 60-9). Titf1-null mice die at birth due to profoundly hypoplastic lungs (Kimura et al., (1996) Genes Dev. 10, 60-9) in addition to other defects, including agenesis of the thyroid and pituitary, and severe malformation of hypothalamus. TITF1 expression appears in the ventral wall of the anterior foregut, an emergence of the lung primordium at E9.5 (Minoo et al., (1999) Dev. Biol. 209, 60-71). TITF1 expression continues in the epithelial cells during lung development and throughout adulthood, at which time, expression is confined to epithelial type II cells (Yuan et al., (2000) Dev. Dyn. 217, 180-90). Titf1-null lungs exhibit only sac-like structures with rudimentary bronchi (Kimura et al., (1996) Genes Dev. 10, 60-9) and do not develop beyond the stage of main bronchi (Minoo et al., (1999) Dev. Biol. 209, 60-71; Yuan et al., (2000) Dev. Dyn. 217, 180-90). The downstream targets for TITF1 that cause this defect are not known.
SCGB3A2, which was originally named uteroglobin related protein 1 (UGRP1), is a member of the uteroglobin/Clara cell secretory protein (UG/CCSP) gene superfamily of secretory proteins, officially named secretoglobin (SCGB) (Klug et al., (2000) Ann. N.Y. Acad. Sci. 923, 348-54). The prototypical protein of this gene superfamily. SCGB1A1 was proposed as a novel cytokine (Mukherjee et al., (1999) Cell Mol Life Sci 55, 771-87). SCGB3A2 was identified as a downstream target for TITF1 in lung using suppressive subtractive library screening of mRNAs isolated from lungs of Titf1-null versus wild-type mice (Niimi et al., (2001) Mol. Endocrinol. 15, 2021-2036), and is predominantly expressed in the epithelial cells of trachea, bronchus, and bronchioles (Niimi et al., (2001) Mol. Endocrinol. 15, 2021-2036).
SCGB3A2 expression is directly regulated by binding of TITF1 to sites located in the promoter of the SCGB3A2 gene (Niimi et al., (2001) Mol. Endocrinol. 15, 2021-2036). Recently, SCGB3A2 was demonstrated to suppress allergen-induced lung inflammation using a mouse model for allergic airway inflammation (Chiba et al., (2006) Am. J. Respir. Crit. Care Med; 173(9):958-64). MARCO, a macrophage scavenger receptor with collagenous structure that is expressed in lung alveolar macrophages and is involved in pulmonary inflammation, was identified as a receptor for SCGB3A2 (Bin et al., (2003) J. Immunol. 171, 924-30). Low SCGB3A2 expression was detected in E12.5 mouse lungs, which markedly increased by E16.5 as determined by RT-PCR (Niimi et al., (2001) Mol. Endocrinol. 15, 2021-2036).
Prior to the present disclosure, the function of SCGB3A2 in lung development had not been determined. As disclosed herein, the arrest of branching and development found in Titf1-null lungs is due to the deficiency of SCGB3A2 expression in these lungs. Indeed, SCGB3A2 exhibits growth factor activity, promoting branching and/or proliferation of Titf1-null lungs ex vivo and in vivo. Rad23b, a downstream target of SCGB3A2 and a component of the DNA damage sensor, mediates this growth factor activity in response to SCGB3A2.
Disclosed herein is the surprising discovery that the secretory protein SCGB3A2 has both growth factor and anti-apoptotic activities, in addition to its previously-known anti-inflammatory activities. In one embodiment of the disclosure, the growth factor activity of SCGB3A2 is exploited for treating and/or inhibiting the development of neonatal respiratory distress syndrome. The efficacy of this method was surprising at least because the MARCO receptor previously was believed to mediate SCGB3A2's effects, and MARCO is expressed only at very low levels in fetal lung. (See, for instance, Elomaa et al., (1995) Cell 80(4):603-609). Thus, prior to the present disclosure that SCGB3A2 also has growth factor activity that is mediated by a receptor other than MARCO, SCGB3A2 would have been expected to have little if any effect on fetal lung tissue.
Neonatal respiratory distress (also called respiratory distress of prematurity and respiratory distress syndrome (RDS)), results from a lack of pulmonary surfactant, a substance that helps the lung's alveoli extract carbon dioxide from the blood and replace it with oxygen. The surfactant prevents the lung's alveoli from collapsing and helps keep them properly inflated by reducing their surface tension. The absence of surfactant prevents the alveoli from functioning properly. RDS primarily affects premature infants, but also is more frequent in infants of diabetic mothers and in the second-born of premature twins.
A human delivery is considered premature when it occurs before 37 weeks of gestation. Although respiratory distress syndrome occurs even in a full-term infant, infants born at about 32 weeks or less gestation are at a greatly increased risk of developing respiratory distress syndrome. The risk of neonatal respiratory distress increases with increasing prematurity, and nearly all fetuses at 24 weeks' gestation or less will suffer from the condition. Most fetuses at 24 weeks or less have not yet developed alveoli, so approximately half of fetuses born at this stage will die, even if treated with surfactant therapy and ventilation. Similar gestational dates can be extrapolated for other species, for example, a mouse gestation generally is approximately 19 to 20 days long, and mice born at E17.5 generally exhibit respiratory distress.
Neonatal respiratory distress syndrome is ameliorated or even prevented entirely if mothers at risk for premature delivery receive SCGB3A2. For example, in some embodiments, SCGB3A2 is administered to the mother if delivery is expected within about two weeks, about one week, or even less than a week, for instance within about three days, about two days, about one day, or even if delivery is expected within a matter of hours. Administration of SCGB3A2 to a gestational mother speeds the development of the fetal lungs and hastens their production of surfactant.
Preterm delivery can be predicted when a mother is exhibiting signs of labor, for instance contractions, cervical dilation and/or thinning, or rupture of the amniotic membrane. In addition, the fetal fibronectin test can help predict whether a woman is likely to give birth within the next 7-14 days. A negative result on the fetal fibronectin test means it is highly unlikely that birth will occur within that time frame. A positive result indicates that premature birth is more likely.
If preterm labor is suspected or anticipated, lung maturity of the fetus can be assessed with various tests carried out on the amniotic fluid. For very premature deliveries, or when there is not sufficient time to carry out a test, SCGB3A2 is administered to the mother without first testing the fetal lung maturity. In pregnancies of greater than about 30 weeks, or in a fetus of any gestational age wherein the lung maturity is uncertain, the fetal lung maturity may be tested by sampling the amount of surfactant in the amniotic fluid, obtained by inserting a needle through the mother's abdomen and uterus. Some maternal conditions, such as diabetes, can impact fetal lung maturity such that surfactant production is insufficient, even at 40 weeks' gestation. The ‘maturity level’ of fetal lungs is expressed as the lecithin-sphingomyelin (or “L/S”) ratio. If this ratio is less than two, the fetal lungs may be surfactant deficient, and SCGB3A2 is administered. In some cases, SCGB3A2 is administered together with a glucocorticoid, which also aids in surfactant production.
Administration to subjects in utero is normally accomplished by transplacental administration or amniotic administration. For transplacental administration, the SCGB3A2 is administered to the mother parenterally, for instance by intravenous, intraperitoneal, intramuscular, intra-arterial, or subcutaneous injection or infusion, or by topical administration. In some instances, maternal administration is by inhalation. Generally, however, intravenous administration is appropriate.
An effective amount of SCGB3A2 is administered in a single dose, or in multiple doses, for example weekly, daily, or multiple times daily, during a course of treatment. In one embodiment, a therapeutically effective amount of a SCGB3A2 is administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time. In pulse doses, a bolus administration of a SCGB3A2 is provided, followed by a time period wherein no SCGB3A2 is administered to the subject, followed by a second (and optionally subsequent) bolus administration. In specific, non-limiting examples, pulse doses of SCGB3A2 are administered during the course of a day, or during the course of a week or more, for instance, from the onset of signs of premature labor until delivery.
In other embodiments, SCGB3A2 is used to treat neonatal respiratory distress postnatally. SCGB3A2 is administered to a neonate parenterally (for example, intravenously, intramuscularly, intraperitoneally, intra-arterially, or subcutaneously), or directly to the lungs by inhalation or by endotracheal tube. By way of example, one method of administration to the lungs is by inhalation through the use of a nebulizer or inhaler. For example, the SCGB3A2 is formulated in an aerosol or particulate and drawn into the lungs using a standard nebulizer well known to those skilled in the art. In addition to SCGB3A2, oxygen is given with a small amount of continuous positive airway pressure (“CPAP”) in some embodiments, and intravenous fluids are administered to stabilize the blood sugar, blood salts, and blood pressure.
Alternatively, or if the infant's condition worsens, an endotracheal tube (breathing tube) is inserted into the trachea and intermittent breaths are given by a mechanical device. SCGB3A2 is administered via the endotracheal tube, and in some embodiments, an exogenous preparation of surfactant, either synthetic or extracted from animal lungs, is also delivered into the lungs in conjunction with the SCGB3A2. Response to treatment is measured by monitoring pulmonary function by methods known to those of skill in the art. For example, various measurable parameters of lung function can be studied before, during, or after treatment. Pulmonary function can be monitored by testing any of several physically measurable operations of a lung including, but not limited to, inspiratory flow rate, expiratory flow rate, lung volume, and oxygen saturation. An increase in one or more of these parameters indicates efficacy of the SCGB3A2 treatment.
For both prenatal and postnatal administration of SCGB3A2, an effective dose ranges from about 0.1 mg/kg to about 100 mg/kg of body weight. In one specific, non-limiting example, an effective dose is from about 1 mg/kg to about 20 mg/kg, or in even more particular examples, from about 5 mg/kg to about 10 mg/kg of body weight.
Also disclosed herein is the surprising discovery that the secretory protein SCGB3A2 promotes lung development. In one embodiment of the disclosure, the growth factory activity of SCGB3A2 is exploited for the promotion of lung development in a subject in need thereof. A subject may be in need of lung development, for instance, because they have or are at risk for developing neonatal respiratory distress. Alternatively, a non-neonatal or adult subject may be in need of lung development because they have suffered lung damage or because they have lost all or part of a lung, or have lost all or part of a lung's capacity or function due to disease, accident or surgery.
Administration to the subject is normally accomplished by either parenteral administration, for instance by intravenous, intraperitoneal, intramuscular, intra-arterial, or subcutaneous injection or infusion, or by inhalation or by endotracheal tube. By way of example, one method of administration to the lungs of an individual is by inhalation through the use of a nebulizer or inhaler. For example, the SCGB3A2 is formulated in an aerosol or particulate and drawn into the lungs using a standard nebulizer well known to those skilled in the art.
In some embodiments, a nanoparticle-based drug delivery system is used for direct pulmonary delivery. (For instance, see Pandey et al., (2003) J. Antimicrobial Chemotherapy 52, 981-986). The small size of the nanoparticles is advantageous for delivery deep into the lungs (see for instance, Jacobs & Muller (2002) Pharmaceutical Research 19, 189-94; Dailey et al., (2003) J. Controlled Release 86, 131-44). In some embodiments, poly(DL-lactide-co-glycolide) (PLG) polymers are chosen as the drug carrier because of their biodegradability and biocompatibility (see, for instance, Anderson & Shive (1997) Advanced Drug Delivery Reviews 28, 5-24). In other embodiments, gelatin-based or polybutylcyanoacrylate-based nanoparticles are used. Such nanoparticles are delivered using a nebulizer, in some embodiments. Nanoparticle-based drug delivery systems are discussed at greater length below, in Example 16.
An effective amount of SCGB3A2 is administered in a single dose, or in multiple doses, for example daily, during a course of treatment. In one embodiment, a therapeutically effective amount of a SCGB3A2 is administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time. Thus, in pulse doses, a bolus administration of a SCGB3A2 is provided, followed by a time period wherein no SCGB3A2 is administered to the subject, followed by a second bolus administration. In specific, non-limiting examples, pulse doses of SCGB3A2 are administered during the course of a day, during the course of a week, or during the course of a month.
Response to treatment is measured by monitoring pulmonary function by methods known to those of skill in the art. For example, various measurable parameters of lung function can be studied before, during, or after treatment. Pulmonary function can be monitored by testing any of several physically measurable operations of a lung including, but not limited to, inspiratory flow rate, expiratory flow rate, lung volume, and oxygen saturation. An increase in one or more of these parameters indicates efficacy of the SCGB3A2 treatment.
The methods of measuring pulmonary function most commonly employed in clinical practice involve timed measurement of inspiratory and expiratory maneuvers to measure specific parameters. For example, forced vital capacity (FVC) measures the total volume in liters exhaled by a patient forcefully from a deep initial inspiration. This parameter, when evaluated in conjunction with the forced expired volume in one second (FEV1), allows bronchoconstriction to be quantitatively evaluated. An increase in FVC or FEV1 reflects a decrease in bronchoconstriction, and indicates that SCGB3A2 therapy is effective.
A concern with forced vital capacity determination is that the forced vital capacity maneuver (for instance, forced exhalation from maximum inspiration to maximum expiration) is largely technique-dependent. In other words, a given subject may produce different FVC values during a sequence of consecutive FVC maneuvers. The FEF 25-75 or forced expiratory flow determined over the midportion of a forced exhalation maneuver tends to be less technique dependent than the FVC. Similarly, the FEV1 tends to be less technique-dependent than FVC. Thus, an increase in the FEF 25-75 or FEV1 reflects a decrease in bronchoconstriction, and indicates that SCGB3A2 therapy is effective.
In addition to measuring volumes of exhaled air as indices of pulmonary function, the flow in liters per minute measured over differing portions of the expiratory cycle can be useful in determining the status of a patient's pulmonary function. In particular, the peak expiratory flow, taken as the highest airflow rate in liters per minute during a forced maximal exhalation, is well correlated with overall pulmonary function in a patient with pulmonary fibrosis and other respiratory diseases. Thus, an increase in the peak expiratory flow following administration of SCGB3A2 indicates that the therapy is effective.
An effective dose ranges from about 0.1 mg/kg to about 100 mg/kg of body weight. In one specific, non-limiting example, an effective dose is from about 1 mg/kg to about 20 mg/kg, or in even more particular examples, from about 5 mg/kg to about 10 mg/kg of body weight. In some embodiments, the SCGB3A2 is administered to the subject in combination with one or more other drugs, such as a bronchodilator, an expectorant, or a steroid.
The effect of SCGB3A2 on bleomycin-induced DNA damage was examined using primary murine fetal lung mesenchymal cells in vitro and a mouse model for bleomycin-induced fibrosis in vivo. In both cases, SCGB3A2 repaired or suppressed bleomycin-induced DNA damage/fibrosis when given together with or prior to bleomycin treatment, respectively. In addition, SCGB3A2 repaired or suppressed bleomycin-induced fibrosis when administered up to two, three, or even four weeks after the commencement of bleomycin treatment. Thus, SCGB3A2 is useful for treating, reducing, and preventing the lung damage caused by bleomycin and other cytotoxic antibiotic antineoplastic agents.
Thus, in yet another embodiment of the disclosure, the anti-apoptotic activity of SCGB3A2 is exploited for the prevention or reduction or reversal (repair) of lung damage in a subject treated with an anti-cancer agent such as a cytotoxic antibiotic. Administration to such a subject is normally accomplished by parenteral administration, for instance by intravenous, intraperitoneal, intramuscular, intra-arterial, or subcutaneous injection or infusion, or by inhalation or by endotracheal tube. By way of example, one method of administration to the lungs of an individual is by inhalation through the use of a nebulizer or inhaler. For example, the SCGB3A2 is formulated in an aerosol or particulate and drawn into the lungs using a standard nebulizer well known to those skilled in the art.
In some embodiments, a nanoparticle-based drug delivery system is used for direct pulmonary delivery. Such nanoparticles are, in some embodiments, poly(DL-lactide-co-glycolide)-based, gelatin-based, or polybutylacrylate-based nanoparticles. Nanoparticle delivery systems are described in greater detail below in Example 16.
As described above, an effective amount of SCGB3A2 is administered in a single dose, or in multiple doses, for example daily, during a course of treatment, or as a single pulse dose, as a bolus dose, or as pulse doses administered over time.
Response to treatment is measured by monitoring pulmonary function by methods known to those of skill in the art. For example, various measurable parameters of lung function can be studied before, during, or after treatment. Pulmonary function can be monitored by testing any of several physically measurable operations of a lung including, but not limited to, inspiratory flow rate, expiratory flow rate, lung volume, and oxygen saturation. An increase in one or more of these parameters indicates efficacy of the SCGB3A2 treatment. These methods and their relative advantages and disadvantages are described elsewhere in greater detail.
An effective dose ranges from about 0.1 mg/kg to about 100 mg/kg of body weight. In one specific, non-limiting example, an effective dose is from about 1 mg/kg to about 20 mg/kg, or in even more particular examples, from about 5 mg/kg to about 10 mg/kg of body weight, based on efficacy. In some embodiments, the SCGB3A2 is administered to the subject in combination with one or more other drugs, such as one or more anti-cancer agents, bronchodilators, expectorants, or steroids.
In some embodiments, SCGB3A2 is used in vitro as a growth factor and/or anti-apoptotic agent, as well as in the study of lung development and the mechanisms of drug-induced DNA damage. For example, recombinant SCGB3A2 is useful for the study of the mechanisms of lung branching morphogenesis, as well as SCGB3A2's anti-apoptotic function against DNA damage caused by bleomycin.
Lung development is regulated by many transcription factors including the zinc finger transcription factors Gli1, 2 and 3. TITF1/NKX2.1, FOXA2/HNF-3β and GATA-6 (see, for instance, Hui et al. (1998) Dev. Biol. 162: 402-413; Motoyama et al. (1998) Nat. Genet. 20: 54-57; Ang & Rossant, (1994) Cell 78: 561-574; Kimura et al. (1996) Genes Dev. 10: 60-69; Perl & Whitsett (1999) Clin. Genet. 56: 14-27; Liu et al. (2002) Am. J. Physiol. Lung Cell. Mol. Physiol. 283: L468-475). Further, several growth factors and morphogens such as fibroblast growth factor (FGF) 10, sonic hedge hog (SHH) and bone morphologic protein (BMP) 4 have been shown to play a role in lung development (Bellusci et al. (1996) Development 122: 1693-170; Litingtung et al. (1998) Nat. Genet. 20: 58-61; Perl & Whitsett (1999) Clin. Genet. 56: 14-27; Sekine et al. (1999) Nat. Genet. 21: 138-141; Cardoso (2001) Annu. Rev. Physiol. 63: 471-494). However, the mechanisms how the activities of these factors relate to each other in promoting branching morphogenesis are not fully understood.
Since SCGB3A2 also influences lung branching morphogenesis (as described herein), understanding how SCGB3A2 affects expression of other factors and participates branching morphogenesis provides a clearer view of the mechanisms of lung branching morphogenesis. For instance, in embryonic lung organ cultures, addition of SCGB3A2 into the media increased mesenchymal cells and the expression levels of FGFs, when examined by RT-PCR.
In order to elucidate the exact location of increased expression and the effects of SCGB3A2 on the expression levels and patterns of other growth factors such as SHH and BMP, for example, various amount of recombinant SCGB3A2 is added to the media of cultures of embryonic lung organ, lung primary cells or cell lines. The levels and/or locations of expression of other growth factors are examined and compared in the presence and absence of SCGB3A2 by quantitative PCR, in situ hybridization, and/or immunohistochemistry. Further, in some embodiments, recombinant SCGB3A2 is bound to beads and placed onto a portion of lung tissue in organ culture. This is particularly useful for elucidating and clarifying the effects of SCGB3A2 on lung branching because the technique restricts the effect of SCGB3A2 to a very small area, compared to the addition of SCGB3A2 to culture media, which affects the entire organ culture.
The influence of SCGB3A2 on the expression levels and patterns of other factors, and how these factors participate in lung branching morphogenesis, is then demonstrated by morphological observation, whole mount hybridization, in situ hybridization, and/or immunohistochemistry. The use of growth factor-bound beads in the study of lung development has been described for BMP4 and FGF10 (Lebeche et al. (1999) Mech. Dev. 86: 125-136; Weaver et al. (2000) Development 127: 2695-2704).
Similarly, recombinant SCGB3A2 is used to elucidate the anti-apoptotic activity of SCGB3A2 by its addition to culture media of embryonic lung organ, lung primary cells or cell lines together, before or after addition of bleomycin. Tissues and/or cells are subjected to microarray and/or protein array analysis, quantitative PCR, northern and/or Western analysis to determine which anti-apoptotic pathway interacts with bleomycin-induced DNA damage pathway.
In other embodiments, SCGB3A2 is used to treat lung cells or lung tissue in vitro, for instance for transplantation into immature, damaged, or diseased lungs. Once transplanted, these cells and/or tissues are used to rebuild or repair the lung damage in vivo. In one embodiment, the lung cell is a stem cell or progenitor cell. The subject of lung epithelial stem cells has been reviewed comprehensively (see, for instance, Rawlins & Hogan (2006) Development 133, 2455-2465; Neuringer & Randall (2004) Respir. Res. 5,6; Neuringer & Randall (2006) Monaldi Arch. Chest Dis. 65, 47-51). Both basal and columnar cells have been shown to reconstitute a complete lung epithelium in an in vivo model of denuded tracheas (see, for instance, Liu et al. (1994) Am. J. Physiol. 266, L296-L307; Avril-Delplanque et al. (2005) Stem Cells 23, 992-1001), and in some species, basal tracheal cells have the ability to form large differentiated epithelial colonies (see, for instance. Hong et al. (2004) Am. J. Physiol. 164, L631-L649). Additionally, Clara cells may be considered stem or progenitor cells because they proliferate to restore the bronchiolar epithelium following injury by oxidant gases (see, for instance, Evans et al. (1986) Am. J. Pathol. 123, 126-133). In general, the lung cells are isolated from a donor airway epithelium, and are dissociated and seeded onto membranes and cultured at the air-liquid interface (see, for instance, Schoch et al. (2004) Am. J. Physiol. Lung Cell Mol. Physiol. 286, L631-L642) in the presence of about 10 ng-10 mg/ml SCGB3A2.
In other embodiments, lung tissue explants are grown in the presence of about 10 ng-10 mg/ml SCGB3A2 in order to prepare the lung tissue for transplantation into an area of diseased or damaged lung tissue. Briefly, in one embodiment, lung tissue is isolated from a donor and cultured in DMEM/F12 containing 10% FBS on a 0.4 μm pore membrane (for instance, Millipore Corporation, Billerica, Mass.), which is placed on the top of steel wire mesh in an organ culture dish (for instance, Becton Dickinson, Franklin Lake, N.J.). Lung explants are then incubated with SCGB3A2 and cultured for two to four days or more at 37° C. in a 5% humidified 95% CO2 air incubator. Every day or every two days, media and the additives (SCGB3A2) are replaced. Once the lung explants have reached the desired size and/or maturity, they are transplanted into an area of diseased or damaged lung.
In other embodiments, lung cells are grown on poly-DL-lactic acid scaffolds in order to engineer lung tissue for transplant (see, for instance, Lin et al. (2006) J. Biomaterials Applications 21, 109-118). Poly-DL-lactic acid (PDLLA) is a well-known super-high molecular-weight acid that has good biocompatibility and degrades following in vivo implantation (see, for instance, Lee & Gardella (2002) Analytical and Bioanalytical Chem., 373(7): 526-537). It has been previously shown that PDLLA, alone or as a composite, supports the growth of osteoblasts, chondrocytes, and lung carcinoma cells (see, for instance, Roether et al. (2002) Biomaterials, 23(18): 387-392; Blaker et al., (2003) J. Biomed. Mater. Res., 67A(4): 1401-1406; Verrier et al. (2004) Biomaterials, 25(15): 3013-3017; Maquet et al., (2004) Biomaterials, 25(18): 4185-4189). These features, plus the intrinsic adequate elastic properties and flexibility of PDLLA, make this biodegradable polymer suitable for lung tissue engineering (see, for instance, Lin et al. (2006) J. Biomaterials Applications 21, 109-118). Methods of culturing lung cells on PDDLA membranes are discussed at greater length in Example 17, below.
SCGB3A2 may be formulated in a variety of ways, depending in part on the type of disease or condition to be treated. Pharmaceutical compositions are provided for both inhalational use and for systemic use, by way of example. The disclosure includes within its scope pharmaceutical compositions comprising SCGB3A2 formulated (or an equivalent thereof) for use in human or veterinary medicine. While SCGB3A2 will typically be used to treat human subjects, it also may be used to treat similar or identical diseases in other vertebrates, such other primates, sheep, chimpanzees, mice, dogs, cats, horses, and cows.
Pharmaceutical compositions that include SCGB3A2 as an active ingredient, or that include both SCGB3A2 and an additional respiratory agent as active ingredients, may be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. Additional active ingredients include, for example, anti-infective agents, anti-inflammatory agents, bronchodilators, enzymes, expectorants, steroids, and anti-cancer agents. A suitable administration format may best be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988.
The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, inhalational formulations are employed in some embodiments. Inhalational preparations include aerosols, particulates, and the like. In general, the goal for particle size for inhalation is about 1 μm or less in order that the pharmaceutical reach the alveolar region of the lung for absorption. Actual methods of preparing such dosage forms are known, or will be apparent, to those of ordinary skill in the art.
The compositions or pharmaceutical compositions can be administered by any route, including parenteral administration, for example, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intraperitoneal, or intramuscular injection or infusion, or by pulmonary inhalation. When SCGB3A2 is provided as parenteral compositions, for instance for injection or infusion, it is generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers. A form of repository or “depot” slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.
For administration by inhalation, the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for instance, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
In some embodiments, a nanoparticle-based drug delivery system is used for direct pulmonary delivery. For instance, poly(DL-lactide-co-glycolide) (PLG) polymers are chosen as the drug carrier because of their biodegradability and biocompatibility (see, for instance, Anderson & Shive (1997) Advanced Drug Delivery Reviews 28, 5-24). In some examples, PLG nanoparticles are prepared using a multiple emulsion technique described by Lamprecht et al. ((1999) International Journal of Pharmaceutics 184, 97-105). The PLG nanoparticles encapsulate the SCGB3A2, and the nanoparticles are administered using a nebulizer.
In other embodiments, the nanoparticles are made from mannitol, lactose, gelatin, or polybutylcyanoacrylate (see, for instance, Sham et al. (2004) International Journal of Pharmaceutics 269, 457-467). To prepare inhalable nanoparticle powders, spray-drying is a commonly practiced method (see, for instance, Bosquillon et al. (2001) J. Control. Release 70, 329-339; Mackin et al. (1997) Pharm. Sci. 3, 583-586; Vanbever et al. (1999) Pharm. Res. 16, 1735-1742). Exemplary methods for the preparation of gelatin nanoparticles can be found in Sham et al. (2004) Int. J. Pharma. 269, 457-467 and Coester et al. (2000) J. Microencapsul. 17, 187-193, and are described below in Example 16.
Without further elaboration, it is believed that one skilled in the art can, using this description, utilize the present discoveries to their fullest extent. The following examples are illustrative only, and not limiting of the disclosure in any way whatsoever.
This Example illustrates specific methods used to carry out the experiments described in Examples 2-14, below.
Two kinds of recombinant mouse SCGB3A2 protein were used that were obtained using a bacterial expression plasmid pET32a-Trx (thioredoxin)-His (histidine)-SCGB3A2 (pET32a from Novagen, San Diego, Calif.) and pDest-544-His6-NusA (N utilization substance protein A)-TEV (Tobacco Etch Virus)-SCGB3A2. In these expression plasmids, a mouse SCGB3A2 cDNA sequence excluding the region encoding the signal peptide (from +155 to +443) was used. Trx-His-tagged recombinant SCGB3A2 protein was produced in BL21 by inducing with isopropyl-β-D-thiogalactopyranosid (IPTG) and was purified by a Ni-NTA Spin Columns (Qiagen, Valencia, Calif.) or TALON Superflow Metal Affinity Resin (BD Biosciences, Palo Alto, Calif.). His6-NusA-TEV-SCGB3A2 protein was produced in Rosetta (DE3) cells (Novagen), which was then subjected to extensive purification steps including TEV protease digestion. The Trx-His-recombinant SCGB32 was used for all ex vivo and cell culture studies whereas highly purified tag-free, endotoxin-free (endotoxin level 0.2 EU/mg) SCGB3A2 was used for all animal studies. Mice received intravenous injection of up to total of 200 μg of SCGB3A2 via the tail vein (less than 5 EU/kg of the maximum endotoxin allowed). All animal studies were carried out in accordance with the Using Animals in Intramural Research Guidelines (NIH Animal Research Advisory Committee, NIH, Bethesda, Md.) after approval by the NCI Animal Care and Use Committee.
Fetal lungs were isolated from pregnant female mice at E11.5-12.0 or E16.5. Lungs were cultured in DMEM/F12 containing 10% FBS on a 0.4 μm pore membrane (Millipore Corporation, Billerica, Mass.), which was placed on the top of steel wire mesh in an organ culture dish (Becton Dickinson, Franklin Lake, N.J.). Lungs were incubated with various additives as indicated, or transfected with Rad23b or negative siRNA probe using HiPerFect Transfection Reagent (Qiagen, Valencia. Calif.), and cultured for two to four days at 37° C. in a 5% humidified 95% CO2 air incubator. Every two days, media and the additives were changed. Experiments were performed using more than three lungs per condition per a given occasion, and were repeated 3-10 times.
Rad23b siRNA Probes:
Rad23b siRNA probes1 and 2 have the following sequences: Probe 1,5′-GUA GCA GGU CAG AAG UUA A-3′ (sense; SEQ ID NO: 1) and 5′-UUA ACU UCU GAC CUG CUA C-3′ (antisense; SEQ ID NO: 2); Probe 2,5′-GCU UCA CAU UAG UAU GAG A-3′ (sense; SEQ ID NO: 3) and 5′-UCU CAU ACU AAU GUG AAG C-3′ (antisense; SEQ ID NO: 4); negative siRNA, 5′-UUC UCC GAA CGU GUC ACG U-3′ (sense; SEQ ID NO: 5) and 5′-ACG UGA CAC GUU CGG AGA A-3′ (antisense; SEQ ID NO: 6).
Lungs were fixed in 4% paraformaldehyde (PFA) overnight at 4° C. and embedded in paraffin or O.C.T. Compound (SAKURA, Torrance, Calif.). Immunohistochemistry and in situ hybridization were carried out as previously described (Chiba et al., (2006) Am. J. Respir. Crit. Care Med May 1; 173(9):958-64; Srisodsai et al., (2004) J. Biol. Chem. 279, 54358-54368) using 4 μm sections. Antibodies used for immunocytochemistry and immunohistochemistry are as follows: anti-histidine (Penta-His antibody, QIAGEN, Valencia, Calif.), anti-thioredoxin (Invitrogen, Carlsbad, Calif.), anti-MARCO (Serotec, Raleigh, N.C.), anti-TITF1 (DAKO, Carpinteria, Calif.), anti-phospho-histone H3 (Upstate, Lake Placid, N.Y.), and anti-Ki-67 (GeneTex, San Antonio, Tex.). Anti-SCGB3A2 antibody was as previously described (Niimi et al., (2001)Mol. Endocrinol. 15, 2021-2036). Cells for positive immunostaining were counted using five areas each (×400 magnification) randomly chosen, from three sections of in vivo tissues and five sections of ex vivo tissues. The number obtained was expressed as a percentage of the total cells.
The E17.5 pups from PBS, 100 μg SCGB3A2, or 200 μg SCGB3A2-treated groups were subjected to measurement of breathing scores. The pups removed from the mothers were placed on moistened filter paper at 37° C. Each pup's breathing motion was observed for two minutes by two independent investigators. Scoring assignment was carried out according to the criteria described by Ozdemir et al. (Pediatr. Res. 53, 98-103 (2003)) as follows: respiratory pattern 0=no breathing; 1=gasping; 2=gasping/labored breathing; 3=labored breathing; 4=labored breathing/unlabored breathing; 5=unlabored breathing. Gasping was characterized as isolated breathing with the mouth opened and the neck extended. Labored breathing involved abdominal and extremity movement, whereas unlabored breathing required chest motion only (Ozdemir et al. (2003) Pediatr. Res. 53, 98-103).
RT-PCR and Quantitative PCR Analysis
Quantitative RT-PCR was performed by SYBR Green master mixture and analyzed with ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, Calif.). The following primers were used for both RT-PCR and quantitative PCR: Rad23b, (forward) 5′-GGA GGA GAC GGT AAA GGC ATT G-3′ (SEQ ID NO: 7), (reverse) 5′-TGG GGA AGA ACT GAC TGT AGT GGG-3′ (SEQ ID NO: 8); FGF2, (forward) 5′-ATG AAG GAA GAT GGA CGG CTG-3′ (SEQ ID NO: 9), (reverse) 5-CCA GTT CGT TTC AGT GCC ACA-3′ (SEQ ID NO: 10); FGF7, (forward) 5′-AAG ACT GTT CTG TCG CAC CCA-3′ (SEQ ID NO: 11), (reverse) 5′-GCC ACA ATT CCA ACT GCC A-3′ (SEQ ID NO: 12); FGF9, (forward) 5′-CTA CCT CGG CAT GAA CGA GAA-3′ (SEQ ID NO: 13), (reverse) 5′-ATC TCC TTC CGG TGT CCA CAT-3′ (SEQ ID NO: 14); FGF10, (forward) 5′-TTT GGT GTC TTC GTT CCC TGT-3′ (SEQ ID NO: 15), (reverse) 5′-AGG TGA TTG TAG CTC CGC ACA-3′ (SEQ ID NO: 16); 18S, (forward) 5′-CGG CTA CCA CAT CCA AGG AA-3′ (SEQ ID NO: 17), (reverse) 5′-ATTGGAGCTGGAATTACCGC-3′ (SEQ ID NO: 18); SP-A, (forward) 5′-TAA GAA GCC AGA GAA CCA GGT AGG-3′ (SEQ ID NO: 23), (reverse) 5′-CTC AGT GAT GTA AAG TGG ACG AAG G-3′ (SEQ ID NO: 24); SP-D, (forward) 5′-TTT GAG GAT GCC CAG GAG ATG TGC-3′ (SEQ ID NO: 25), (reverse) 5′-AGG AAA GCA GCC TTG TTG TGG-3′ (SEQ ID NO: 26); AQP1, (forward) 5′-GCT CAC CCG CAA CTT CTC AAA C-3′ (SEQ ID NO: 27), (reverse) 5′-TCA GCA TCC AGG TCA TAC TCC TCC-3′ (SEQ ID NO: 28); OB-R, (forward) 5′-AGG AAT CGT TCT GCA AAT CCA-3′ (SEQ ID NO: 29), (reverse) 5′-TAT GCC AGG TTA AGT GCA GCT ATC-3′ (SEQ ID NO: 30).
Unless otherwise specifically noted in particular Examples below, the conditions used for RT-PCR were 94° C., 5 minutes, followed by 94° C. for 15 seconds, 60° C. or 62° C. (for Rad23b) for 15 seconds, 72° C. for 30 seconds, 72° C. for five minutes for 25-28 cycles depending on gene, and for quantitative PCR, 50° C. for 2 minutes, 95° C. for 10 minutes, followed by 95° C. for 15 seconds, 60 or 62° C. (for Rad23b) for 40 seconds for 45 cycles. The standard curve method was used and all data were normalized to 18S rRNA.
Construction of pDest-544-His6-NusA-TEV-SCGB3A2
pDonr223 is a Gateway Donor vector modified from pDonr201 (Invitrogen). In pDonr223, the kanamycin resistance gene is replaced with a gene encoding spectinomycin resistance, and several sequencing primer sites have been added to aid in sequence verification of Entry clones. pDest-544 is a Gateway Destination vector generated by modification of pET-43b (Novagen, Inc), which contains a T7 promoter and an amino-terminal His6-NusA fusion tag. The following oligonucleotides (Operon, Inc) were used in this study: L2575, 5′-GGC GAA AAC CTG TAC TTC CAA GGC ATG CTT CTC ATC AAC CGT CTC CCT GTT GTT GAC-3′ (SEQ ID NO: 19); L2576, 5′-GGG GAC AAC TTT GTA CAA GAA AGT TGG CTA TAC CAG GTG TGA AAG AGC CTC C-3′ (SEQ ID NO: 20); L907, 5′-GGG GAC AAC TTT GTA CAA AAA AGT TGG CGA AAA CCT GTA CTT CCA AGG C-3′ (SEQ ID NO: 21).
A SCGB3A2 cDNA fragment used to construct pDest-544-His6-NusA-TEV-SCGB3A2 was first amplified using 95° C. for two minutes, followed by five cycles of 95° C. for 30 seconds, 55° C. for 45 seconds, and 72° C. for 60 seconds with 200 nM each of primers L2575 (SEQ ID NO: 19) and L2576 (SEQ ID NO: 20), and the original SCGB3A2 cDNA clone (Niimi et al. (2001) Mol. Endocrinol. 15, 2021-2036; clone #12). Following this, 200 nM of adapter primer L907 (SEQ ID NO: 21) was added, and 15 more cycles of amplification were carried out using the same cycling parameters with Platinum Taq HiFidelity (Invitrogen). All cycling was done on an Applied Biosystems 9700 cycler. After cycling, a 72° C., five minute incubation was carried out to extend PCR products, followed by cooling to 4° C. The final PCR product contains the mature form of the SCGB3A2 gene (starting at amino acid 22 of the full-length protein sequence) flanked on the 5′ side with a Gateway attB1 site and Tobacco Etch Virus (TEV) protease cleavage site. The 3′ side contains a Gateway attB2 recombination site. The PCR products were cleaned with the QiaQuick PCR purification kit (Qiagen), and recombined into pDonr223 by Gateway BP recombination (Invitrogen) using the manufacturer's protocols. The subsequent Entry clone was sequence verified, and subcloned by Gateway LR recombination into pDest-544. The final expression clone (3152-X1-544) encodes a protein of the form His6-NusA-TEV-SCGB3A2 mature. The linker between the fusion tag and the SCGB3A2 gene consists of the amino acid sequence ENLYFQG (SEQ ID NO: 22), which is cleaved by Tev protease between the Q and G residues to leave only a single glycine at the aminoterminus of the mature SCGB3A2 gene.
Expression of His6-NusA-TEV-SCGB3A2
The expression clone was transformed into E. coli Rosetta (DE3) cells (Novagen). Five ml of an overnight culture grown at 37° C. in CircleGrow medium (Qbiogene) containing 100 μg/ml ampicillin and 15 μg/ml chloramphenicol were used to innoculate a four-liter baffled shake flask containing one liter of LB medium with the same antibiotics. After incubation at 37° C. at 200 rpm for seven hours, 450 ml of this seed culture (OD600=2.10) were added to a 20 liter New Brunswick Scientific BioFlo IV fermenter containing 15 liters of modified Studier ZYM5052 medium, whose final composition was 1% N-Z-amine AS, 0.5% yeast extract, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 1 mM trisodium citrate, 0.2× trace metals, 2% glycerol, 0.05% glucose, 0.2% α-lactose, 25 mM succinic acid, 100 μg/mL ampicillin, and 15 μg/mL chloramphenicol. This medium corresponds to ZYP5052 with the following adjustments: salts were at half concentration, glycerol was increased from 0.5 to 2% to increase cell yield, and succinic acid was added to stabilize pH. Cells were grown at 37° C. with monitoring (but without control) of pH but without supplemental feeding, and dissolved oxygen was maintained at 40%. After five hours (A600=5.19), the temperature was reduced to 20° C. and growth continued until harvest at 18 hours (stationary phase was reached at 16 hours, final A600=24.0, final pH 5.42). Cells (3.6×105 A600 units, 626 grams wet weight) were harvested and stored at −80° until use.
Non-Denaturing Purification by Immobilized Metal Ion Affinity Chromatography (IMAC)
E. coli cell pastes were resuspended with four volumes of extraction buffer (for a final concentration of 20 mM HEPES, pH 7.3, 100 mM NaCl, 5 mM MgCl2, 5% glycerol, 45 mM imidazole, and Complete protease inhibitor as per the manufacturer's instructions, Roche) per gram wet weight, digested with lysozyme (0.5 mg/ml, Sigma) for 30 minutes on ice and treated with 10 U Benzonase (Novagen)/ml for an additional 20 minutes. The sample was sonicated to lyse the cells using a Branson Digital Sonifier 450 (100% power, 33% duty cycle, for 3×20 sec with a 1 cm tip) and lysis was verified by microscopic examination. The lysate was adjusted to 500 mM NaCl, clarified by centrifugation at 111,000×g for 30 minutes, filtered (0.45 PES membrane) and applied to HisTrap columns (GE Healthcare) equilibrated with extraction buffer in 500 mM NaCl and 45 mM imidazole (binding buffer). The columns were washed with binding buffer to baseline, proteins eluted over 20 column volume (CV) gradients to 400 mM imidazole.
TEV Protease Digestion and Subsequent Purification
The pool created from the IMAC was dialyzed against binding buffer without additional imidazole and treated with TEV protease (3% v/v with a 5 mg/ml lab stock) at room temperature for four hours and then shifted to 16° C. overnight and analyzed by SDS-PAGE. An additional IMAC step similar to the initial IMAC was used to purify the target protein away from the contaminants of the protease digest.
A protein sample was dialyzed against 20 mM HEPES, pH 7.3, 100 mM
NaCl and 5% glycerol and applied to Q Sepharose resin in pre-poured columns (GE Healthcare) using a chromatography workstation (AKTA-Purifier, GE Healthcare) made endotoxin free by cleaning with 0.01% Triton X-114 (Sigma). Proteins were eluted with a 0.1 to 1M NaCl gradient over 20 CV. At each purification step, fractions were analyzed by SDS-PAGE, and tested for endotoxin levels. The final endotoxin levels were determined by Cambrex Bio Science (Walkersville, Md.).
Samples were then dialyzed twice for at least four hours at 4° C. against at least 20 sample volumes of final buffer using 3.5K MWCO Snakeskin (Pierce) dialysis membrane. When necessary, samples were concentrated using 5K MWCO Amicon Ultra filtration devices (Millipore).
5-Bromo-2′-deoxyuridine (BrdU) was directly added to the culture medium of fetal lung primary cells to achieve a final concentration of 10 μM, and cells were incubated for one hour at 37 C.°. Cells were then trypsinized and fixed in 75% ethanol for 24 hours. Fixed cells were treated with 1 ml of 2N HCl containing 0.5% triton X-100 at room temperature for 30 minutes and neutralized with 1 ml of 0.1 M Na2B4O7.10H2O, PH 8.5. The cells were centrifuged and suspended in 1 ml of PBS containing 0.5% Tween 20 and 1% BSA, and incubated for 30 minutes at room temperature with 20 μl of Anti-BrdU FITC (Becton Dickinson, San Jose, Calif.), followed by centrifugation and resuspension in 1 ml of PBS containing 5 μg/ml pf propidium iodide (BD Biosciences Pharmingen, San Diego, Calif.). Measurement of red (DNA content) and green (BrdU) fluorescence was determined by flow cytometry.
Flow Cytometric Analysis
Cells were analyzed using a single laser FACS Calibur cytometer (Becton Dickinson, San Jose, Calif.) with excitation at 488 nm. The data were collected and analyzed employing CellQuest Software. The red fluorescent signals (DNA content) were analyzed on a linear scale and the green signals (BrdU) were analyzed on a logarithmic scale. The data are presented as a percentage of the total population exhibiting the fluorescence signal of interest. For each sample, 10,000 cells were analyzed.
Titf1-null lungs at E16.5 were cultured in an organ culture system with or without 250 ng/ml SCGB3A2 protein for four days. Total RNAs from these lungs were isolated by TRIzol (Invitrogen, Carlsbad, Calif.) and then incubated with DNase I (Ambion, Austin, Tex.) for 20 minutes at 37° C. RNAs were amplified using MessageAmp aRNA Kit (Ambion, Austin, Tex.), and amplified RNAs were reverse-transcribed to label with Cy3 and Cy5 (GE Healthcare Life Sciences, Piscataway, N.J.) using FairPlay Microarray Labeling Kit (Stratagene, La Jolla, Calif.). Ten mouse arrays (42.2 K) obtained from the NCI Microarray Facility were used for this experiment. Experiments and analysis were performed according to the manufacturer's instructions and the protocol of the Center for Cancer Research, NCI.
This Example demonstrates that SCGB3A2 increases branching morphogenesis in fetal lung explant cultures.
SCGB3A2 expression was detected by immunohistochemistry, albeit at low levels in the epithelial cells of E11.5 and 13.5 normal fetal lungs (
After four days of culture, normal fetal lung harvested at E11.5 (
This Example demonstrates that SCGB3A2 promotes branching in lungs from Titf1-null mice and produces a recovery of the epithelial characteristics of Titf1-null mouse trachea and lung similar to those found in wild-type mice.
Titf1-null lungs exhibit vacuolar lobes consisting of rudimentary bronchi with lean mesenchymal layers (
The epithelia of trachea and lung in Titf1-null mice were comprised of one to two layers of columnar epithelial cells covered with flattened epithelial and few ciliated cells (
This Example demonstrates that the phenotypes produced by SCGB3A2-treatment of trachea and lungs from Titf1-null mice are caused by increased cell proliferation.
To examine whether increased proliferation is responsible for the phenotypes observed in the Titf1-null lungs and trachea by SCGB3A2 treatment, the expression of phosphorylated histone H3 as a mitosis marker and Ki-67 as a proliferation marker, was examined by immunohistochemistry using Titf1-null mice with and without SCGB3A2. In ex vivo cultured Titf1-null lungs as well as lung and trachea of Titf1-null mice, both phosphorylated histone H3 and Ki-67 were very weakly expressed without SCGB3A2 in both epithelial and mesenchymal cells (see, e.g.,
Further, the effect of SCGB3A2 on cell proliferation was accurately determined by BrdU incorporation into primary mesenchymal cells prepared from wild-type mouse fetal lung. A statistically significant increase was obtained, indicating that SCGB3A2 enhanced cell proliferation (
Since SCGB3A2 treatment increased mesenchymal cells in fetal lungs, it was interesting to determine the expression levels of several FGFs, the growth factor mainly expressed in mesenchymes and known to be involved in lung development. Among FGFs analyzed by RT-PCR using RNAs isolated from ex vivo cultured Titf1-null fetal lungs, FGF7 expression was dramatically increased, while FGF2 and FGF9 were minimally increased upon SCGB3A2 treatment (
This Example demonstrates that the known receptor for SCGB3A2. MARCO, is not responsible for the effects of SCGB3A2 on fetal lungs, and that an independent SCGB3A2-specific receptor likely exists in fetal lungs, especially in mesenchyme.
MARCO is the known receptor for SCGB3A2 (Budinger et al., (2006) Proc. Natl. Acad. Sci. U.S.A. 103:4604-4609). In order to determine whether a receptor for SCGB3A2 other than MARCO is present in fetal lung, and where it might be localized, wild-type fetal lung primary epithelial and mesenchymal cells were treated with histidine and thioredoxin tag-containing SCGB3A2, and were then subjected to immunocytochemistry using anti-histidine and anti-thioredoxin antibodies (
This Example illustrates the fact that Rad23b is a downstream target of SCGB3A2 that is involved in SCGB3A2's proliferative effects.
In order to identify genes that are controlled by SCGB3A2 and are responsible for branching, mouse DNA microarray analysis was carried out using Titf1-null lungs with and without SCGB3A2 treatment. Over eighty SCGB3A2-up-regulated genes were selected for further examination for the level and localization of expression by quantitative PCR and in situ hybridization. The focus was on genes localized to the mesenchyme, in which a SCGB3A2-specific receptor is likely to be present.
Expression of one gene, Rad23b, was always up-regulated by SCGB3A2 in all microarray analyses carried out (n=5), and the representative score was 2.4892±0.242048. Rad23b expression was increased 4.73-fold in the ex vivo Titf1-null lungs after SCGB3A2 stimulation as determined by quantitative PCR.
Rad23b is one of the two Mus musculus homologs of the Saccharomyces cerevisiae DNA repair protein RAD23, and is tightly complexed with xeroderma pigmentosum group C(XPC), serving as a primary DNA damage sensor (Nord et al., 2000 Ann N Y Acad Sci 923:154-165). In E13.5 normal fetal lung, in situ hybridization demonstrated that Rad23b mRNA was mainly detected in mesenchymal cells (
This Example demonstrates phenotypic effects of knocking down Rad23b expression.
In order to confirm that Rad23b is a SCGB3A2 downstream target responsible for lung development, the effect of knocking down Rad23b mRNA using specific siRNA on lung branching morphogenesis was examined in ex vivo lung culture studies. First, Rad23b specific siRNA probes were transfected into primary mesenchymal cells prepared from wild-type fetal lungs. Among four probes examined, two (designated 1 and 2) markedly decreased Rad23b mRNA levels to 2.8% and 6.2%, respectively relative to control non-siRNA transfected cells (
Next, Rad23b siRNA probe1 was used for ex vivo culture studies of fetal lungs (
This Example describes SCGB3A2's growth factor activities and effects on lung development.
As described herein, SCGB3A2 promotes branching and proliferation of fetal lung, mediated through the downstream target Rad23b. This is the first report describing SCGB3A2 as a growth factor in addition to an anti-inflammatory agent, as was demonstrated using a mouse model for allergic airway inflammation (Chiba et al., (2006) Am. J. Respir. Crit. Care Med May 1; 173(9):958-64). This growth factor activity of SCGB3A2 seems to be in accordance with the fact that expression of SCGB3A2 is found at E11.5 and greatly increases by E16.5, during the period when lung undergoes dramatic morphological changes.
SCGB3A2 was originally identified as a downstream target for TITF1 in lung using suppressive subtractive hybridization between wild-type and Titf1-null lungs (Niimi et al., (2001) Mol. Endocrinol. 15, 2021-2036). SCGB3A2 expression was directly regulated by TITF1 through its binding to specific elements located in the promoter of SCGB3A2 gene (Niimi et al., (2001) Mol. Endocrinol. 15, 2021-2036). SCGB3A2 belongs to the secretoglobin (SCGB) gene superfamily of small secretory proteins. The prototypical protein of the SCGB gene superfamily, SCGB1A1, was proposed as a novel cytokine (Mukherjee et al., (1999) Cell Mol Life Sci 55, 771-87). SCGB1A1 is highly expressed in the airways after E16.5 throughout adulthood (Peri et al., (1995) J Clin Invest 96:343-353; Singh & Katyal (2000) Ann N Y Acad Sci 923:43-58) and is known to exhibit an anti-inflammatory function (Ye et al., (2004) Respiration 71:505-510; Zhang et al., (1997) Science 276:1408-1412). In this context, it is interesting to note that Scgb1a1-knockout mice exhibit no abnormalities in their lungs (Warburton et al., (2000)Mech Dev 92:55-81).
Assuming that SCGB3A2 plays a role in lung development, it was interesting to examine whether SCGB3A2 is capable of recovering the development of Titf1-null lungs. As demonstrated herein, the arrest of branching and morphogenesis found in Titf1-null lungs is indeed partly attributed to the deficiency of SCGB3A2. Thus, Titf1-null lung ex vivo cultures exhibited dramatic morphological changes by the addition of SCGB3A2 in the culture medium, presenting pleated and dentate epithelial layers with increased mesenchymes. These morphological changes indicate that SCGB3A2 induced proliferation and invagination, characteristic processes for branching (Minoo et al., (1999) Dev Biol 209:60-71; Kaplan (2000) Mol Genet Metab 71:321-341).
In in vivo experiments, no invagination was observed, which might be due to low concentrations of SCGB3A2 protein that can reach the lung and/or due to space constraint in the thorax. Nevertheless, the morphological changes observed in SCGB3A2-stimulated Titf1-null lungs somewhat resemble those of tracheal development in Drosophila (Minoo et al., (1999) Dev Biol 209:60-71; Kaplan (2000) Mol Genet Metab 71:321-341) in that the epithelia in Titf1-null trachea and lung were almost barren, and after SCGB3A2 treatment, they became composed of ciliated columnar cells including goblet cells in the entire epithelia. Increased expression of proliferation markers, Ki-67 and phosphorylated histone H3, demonstrated that SCGB3A2 induced proliferation in Titf1-null lung. The effect of SCGB3A2 on cell proliferation was further confirmed in wild-type fetal lung primary mesenchymal cells by BrdU incorporation experiments. In support of this, increased expression of FGFs, particularly FGF7, was found in SCGB3A2-treated ex vivo Titf1-null fetal lungs. This further indicates that epithelial cells proliferate in the presence of SCG3A2 through FGF7 by epithelial-mesenchymal interaction (Wallach-Dayan et al., (2006) Am J Physiol Lung Cell Mol Physiol 290:L790-L796; Ng et al., (2002) Mol Cell Biol 22:1233-1245).
MARCO (macrophage scavenger receptor with collagenous structure) was reported as a SCGB3A2 receptor in human (Bin et al. (2003) J. Immunol. 171, 924-30). MARCO is also known as a receptor for lipopolysaccharide and plays a role in inflammation (Budinger et al., (2006) Proc. Natl. Acad. Sci. U.S.A. 103:4604-4609). While the expression of MARCO was found in alveolar macrophages of adult mouse lung by in situ hybridization as expected, the expression was not observed in E13.5 fetal lungs as determined by either in situ hybridization or immunohistochemistry. Further, treatment of wild-type primary fetal lung culture cells with excess SCGB3A2 protein gave positive signals predominantly in the mesenchymal fraction of cells only with anti-histidine and anti-thioredoxin antibodies, but not anti-MARCO antibody, indicating that an unknown SCGB3A2-specific receptor likely exists in fetal lungs, especially in mesenchyme.
DNA microarray analysis was carried out to understand what molecules function through SCGB3A2 signaling in order to promote proliferation and branching in fetal lungs. Among genes up-regulated. Rad23b was selected for further study as a candidate downstream target for SCGB3A2 because Rad23b was mainly expressed in mesenchyme at early embryonic days. Mice lacking the Rad23b gene, its homologue Rad23a, or both genes have been established (Ng et al., (2003) Genes Dev 17:1630-1645; Katiyar et al., (2005) Biochem Biophys Res Commun 337:1296-1300). Interestingly, Rad23b-null mice exhibit impaired embryonic development (Ng et al., (2003) Genes Dev 17:1630-1645). Expression of the human RAD23B gene was found both in cytoplasm and nuclei during G1 phase and mainly in cytoplasm during S phase (Lebeche et al., (1999) Mech Dev 86:125-136).
In the present disclosure, Rad23b expression was observed at E13.5 of normal fetal lungs, which increased by E16.5, coinciding with the stage of SCGB3A2 increase. SCGB3A2 also up-regulated Rad23b expression in Titf1-null lung ex vivo and in vivo. Further, Rad23b siRNA delayed branching morphogenesis when examined by ex vivo organ culture studies; this delay was counteracted by the addition of SCGB3A2. This counteraction, however, was not as effective as those of control or negative siRNA in the presence of SCGB3A2. This could be due to a competition between Rad23b siRNA and SCGB3A2 at the concentrations used.
In conclusion, disclosed herein is the discovery that SCGB3A2 is a growth factor, playing a critical role in branching morphogenesis in fetal lungs, whose activity is mediated through the DNA damage sensor gene Rad23b. SCGB3A2 is one of the major genes responsible for the phenotypes observed in the Titf1-null lungs. This growth factor activity of SCGB3A2 indicates that SCGB3A2 can be used to treat many lung diseases such as asthma, chronic obstructive pulmonary disease, cancer, and neonatal respiratory distress.
This Example provides specific methods used for carrying out studies using SCGB3A2 to prevent or reverse lung damage caused by anti-cancer agents such as bleomycin.
For the bleomycin study, 7-8-week-old C57BL/6 female mice or SCGB3A2 transgenic mice that over-express SCGB3A2 in lung, were intratracheally intubated with bleomycin (8 U/kg of body weight; Sigma-Aldrich, St. Louis, Mo.) or PBS as control, or sham intubated at day 0, and euthanized after 3 weeks. Some mice received SCGB3A2 intravenously for a week starting day 1. All animal studies were carried out in accordance with the Using Animals in Intramural Research Guidelines (NIH Animal Research Advisory Committee, NIH, Bethesda, Md.) after approval by the NCI Animal Care and Use Committee.
Fetal lungs at E16.5 were incubated in DMEM/F12 containing 10% FBS, 1 U/ml Dispase I (Roche Applied Science, Indianapolis, Ind.) and 1,000 U/ml collagenase (Sigma-Aldrich) at 37° C. for 30 minutes with shaking. After incubation, cells were washed three times in DMEM/F12 containing 10% fetal bovine serum (FBS). Cells were then plated onto a 10 cm-plate and incubated for 20 min at 37° C. to separate epithelial cells from mesenchymal cells (Lebeche et al., (1999) Mech Dev 86:125-136). While mesenchymal cells attached to the plates, epithelial cells that remain in the media were transferred to a new plate. Embryonic lung primary culture cells were seeded on a 24-well plate at 3×105 cells/ml for transfection of siRNA or BrdU incorporation. Transfection was carried out using HiPerFect Transfection Reagent (QIAGEN, Valencia, Calif.) according to the manufacturer's protocol.
For TUNEL assay (Promega, San Luis Obispo, Calif. USA) for bleomycin-damaged DNAs, primary fetal lung mesenchymal cells were cultured in LAB-TEK 8-chamber slides (Nalge Nunc International, Rochester, N.Y.) for 44 hours before the addition of bleomycin and harvested 20 hours later. SCGB3A2 was either added together with, or 20 hours earlier than bleomycin addition.
Lungs were fixed in 4% paraformaldehyde (PFA) overnight at 4° C. and embedded in paraffin or O.C.T. Compound (SAKURA, Torrance, Calif.). TUNEL assay was performed to detect apoptotic cells in tissue sections using commercially available kit (Promega, Madison, Wis.). Tissue sections were processed according to the manufacturer's instruction, through which a biotinylated nucleotide was incorporated at the 3′-OH end of fragmented DNAs. Biotin incorporation was detected with HRP conjugated streptavidin using DAB as a chromogen.
Bleomycin is well known for causing DNA damage and apoptosis (Povirk (1996) Mutat Res 355:71-89; Lee et al., (2005) Am J Physiol Lung Cell Mol Physiol 289:L521-528; Wang et al., (2000) Am. J. Physiol Lung Cell Mol Physiol 279:L143-151; Budinger et al., (2006) Proc. Natl. Acad. Sci. U.S.A. 103:4604-4609; Wallach-Dayan et al., (2006) Am J Physiol Lung Cell Mol Physiol 290:L790-L796). Since Rad23b was identified as a component of DNA damage sensor (Sugasawa et al., (1998) Mol Cell 2:223-232), and as described herein SCGB3A2 exerts its proliferation activity through Rad23b, it was proposed to determine if SCGB3A2 may affect the extent of bleomycin-induced DNA damage/apoptosis.
To this end, fetal lung primary mesenchymal cells were treated with bleomycin with and without SCGB3A2 (
This Example demonstrates that SCGB3A2 can be used to inhibit or reduce lung damage caused by anti-cancer agents such as bleomycin.
Bleomycin, when given to mice by intratracheal intubation, produces interstitial pneumonia and pulmonary fibrosis, which is used as a model for studying human interstitial pneumonia and pulmonary fibrosis (Polosukhin et al., (2005) Ultrastruct Pathol 29:53-64; Grande et al., (1998) Scanning Microscopy 12:487-494). In order to examine the effect of SCGB3A2 on bleomycin-induced pulmonary fibrosis, wild-type mice as well as SCGB3A2 transgenic mice over-expressing SCGB3A2 in lung under the promoter of human surfactant protein C gene, were subjected to an experimental model mouse for bleomycin-induced pulmonary fibrosis.
After bleomycin intubation, wild-type mice did not gain weight for several days, and although they gradually recovered, never reached the weights of control group of mice (
Next, SCGB3A2 transgenic mice were given bleomycin, and the changes in body weight and tissue histology were compared with those of SCGB3A2 transgenic and wild-type mice intubated with PBS, and wild-type mice intubated with bleomycin (
Bleomycin is one of many anti-neoplastic agents that are known to induce DNA damage and apoptosis in lung, resulting in pulmonary fibrosis (Povirk (1996) Mutat Res 355:71-89; Lee et al.; (2005) Am J Physiol Lung Cell Mol Physiol 289:L521-528; Wang et al., (2000) Am J Physiol Lung Cell Mol Physiol 279:L143-151; Budinger et al., (2006) Proc. Natl. Acad. Sci. U.S.A. 103:4604-4609; Wallach-Dayan et al., (2006) Am J Physiol Lung Cell Mol Physiol 290:L790-L796). Although the involvement of JUNK-dependent mitochondrial death pathway (Povirk (1996) Mutat Res 355:71-89), caspase-8 and -9 (Wallach-Dayan et al., (2006) Am J Physiol Lung Cell Mol Physiol 290:L790-L796), and Bcl-2 family member Bid (Budinger et al., (2006) Proc. Natl. Acad. Sci. U.S.A. 103:4604-4609) has been suggested, the molecular mechanism for bleomycin-induced apoptosis is poorly understood. As disclosed herein, SCGB3A2 functions as an anti bleomycin-induced apoptosis reagent in vitro, and in a mouse model for pulmonary fibrosis it can repair bleomycin-induced interstitial pneumonia and fibrosis. When SCGB3A2 was given prior to bleomycin or by high levels of SCGB3A2 expression such as found in SCGB3A2 transgenic mice, DNA damage by bleomycin did not occur, indicating that the SCGB3A2 signaling pathway may interact with bleomycin-induced apoptosis pathway. Thus, SCGB3A2 can intervene and/or suppress pulmonary interstitial pneumonia and fibrosis.
This Example provides methods that were used to demonstrate that SCGB3A2 inhibits or repairs existing lung damage caused by bleomycin.
Bronchoalveolar Lavage (BAL) Fluid and Cell Count
BAL fluid was obtained after mice were euthanized, by intratracheal instillation of 1 ml PBS in the lung while it was kept located within the thoracic cavity. The lavage was reinfused in the lung two times before final collection. BAL cells were isolated by centrifugation at 3,000 rpm for 10 minutes, and resuspended in 20 μl PBS. Smear preparation of BAL cells were stained with Diff-Quik Stain Set (Dade Behring Inc. Deerfield, Ill.). Cells in 2.25-6.5 cm2 areas of smear preparation (using 3-5 mice) were counted.
Masson trichrome staining to detect collagen fibers was carried out using Accustain Trichrome Stains (Masson) (Sigma-Aldrich, St. Louis, Mo.) with slight modification.
The effect of SCGB3A2 on bleomycin-induced DNA damage was examined using primary fetal lung mesenchymal cells in vitro and a mouse model for bleomycin-induced fibrosis in vivo. In both cases, SCGB3A2 repaired or suppressed bleomycin-induced DNA damage/fibrosis when given together or prior to bleomycin treatment, respectively. Mice were intratracheally administered PBS or bleomycin; each group was further sub-grouped into those that received intravenous injection of PBS or SCGB3A2 daily for a week, starting at the 14th day of the experiment (total of 4 groups) (
H&E stained whole lung tissues (n=5 in each group) were graded based on the percentage of fibrous area (
Next, inflammatory cells in BAL fluid, which were increased as a result of fibrosis and tissue damage, were counted in all groups of mice (
The present results demonstrate the striking effect of SCGB3A2 on restoring normal histology of lungs that once had fibrosis. Previously, mice treated with SCGB3A2 daily for a week starting one day after bleomycin administration or SCGB3A2 transgenic mice that over-express SCGB3A2 in their airways, did not develop fibrosis after 3 weeks of bleomycin administration. This is likely due to the fact that SCGB3A2 suppresses apoptosis caused by bleomycin. This early phase effect of SCGB3A2, however, appears to be different from the currently observed effect where SCGB3A2 repaired bleomycin-induced lung fibrosis after it has already begun.
The mechanisms for pulmonary fibrosis are not known. It is currently believed that pulmonary fibrosis cannot be reversed once it has begun. However, as disclosed herein, SCGB3A2 can be used to treat pulmonary fibrosis, particularly at the early stages of the disease. Thus, SCGB3A2 may be given to cancer patients who undergo bleomycin chemotherapy in order to reduce side effects and/or block bleomycin-induced fibrosis.
This Example demonstrates that SCGB3A2, when administered to the mother, promotes lung development in pre-term mouse pups. The effect of SCGB3A2 was examined on the late gestational stages of fetal lung development in vivo by injecting highly purified tag-free, endotoxin-free SCGB3A2 daily from E13.5 through E16.5 through the tail vein of pregnant female mice, followed by removal of pups from the mother at E17.5. Pups are usually born at E19.0-20.0.
Pups removed at E17.5 from mothers receiving a total of 100 and 200 μg SCGB3A2 displayed similar body length (to each other), and body and lung weights, which were larger than PBS-treated control pups (
The expression of several genes known to have markedly increased expression towards the end of gestation was examined by qPCR (
Finally, amniotic fluid lipidomes of the SCGB3A2-treated mice were compared with those of immature E17.5 and mature E19.0 controls through a LC-MS-based metabolomic analysis (
Thus, administration of SCGB3A2 to a pregnant female mouse promoted lung development of preterm pups, which exhibited equivalent lung phenotypes to those of matured term pups. No obvious abnormality was noted with any other organs/tissues of preterm pups, or in mothers treated with SCGB3A2. However, body length and weight were slightly increased in SCGB3A2-treated pups, in accordance with the increase of lung weight. Thus, without intending to be bound by theory, SCGB3A2 may be involved in general pathways of growth promotion. Alternatively, there might be a factor(s) that sets a size of the body in order to accommodate developing lung.
Because the limited volume of mouse amniotic fluid available prevented the use of traditional thin-layer chromatography (Grenache & Gronowski, (2006) Clin. Biochem. 39, 1-10), a metabolomic approach that has been adopted in other lipid-related research fields (Griffin & Nicholls (2006) Pharmacogenomics 7, 1095-107) was used to examine and compare the lipid profiles of amniotic fluid from control and SCGB3A2-treated samples. Distinctive grouping of immature and mature amniotic fluid samples as well as the samples from SCGB3A2 treatments demonstrated the phenotyping capability of metabolomics on the fetal lung development based on the lipid species in the amniotic fluid. In conclusion, these results demonstrate that SCGB3A2 is a powerful treatment for and preventative of neonatal respiratory distress syndrome.
This Example provides an exemplary protocol for the treatment of neonatal respiratory distress in humans.
In particular, non-limiting examples, fetal administration is accomplished by transplacental administration, for example by administering the SCGB3A2 to the mother intravenously. Depending on the physical condition of the mother and fetus, and on the length of time available before the anticipated delivery, SCGB3A2 is administered in a dose of from about 0.1 mg/kg to about 100 mg/kg of maternal weight. In one specific, non-limiting example, an effective dose is from about 1 mg/kg to about 20 mg/kg, or in even more particular examples, from about 5 mg/kg to about 10 mg/kg of maternal weight.
The SCGB3A2 can be administered in a single dose, or in multiple doses, for example daily, during a course of treatment. In one embodiment, SCGB3A2 is administered as a single dose of from about 5 mg/kg to about 10 mg/kg of maternal weight. In some instances, this single dose of SCGB3A2 is sufficient to promote lung development such that, following delivery, the infant will have sufficiently mature lungs to breathe independently.
In another embodiment, SCGB3A2 is administered as a series of pulse doses during the course of treatment for as long as delivery can be delayed. For instance, in some embodiments, SCGB3A2 is administered once daily until the time of delivery, or more frequently, for instance twice daily or every 4 or 6 hours, for example.
In some embodiments, SCGB3A2 therapy is combined with glucocorticoid treatment, which also speeds surfactant production. During the course of treatment, lung maturity can continue to be monitored, until such time as the lungs have matured and/or delivery can no longer be delayed. If needed, in some embodiments, the newborn receives ex-utero SCGB3A2 treatment and/or artificial surfactant to further promote lung maturation and function. In many cases, however, in utero treatment with SCB3A2 is sufficient to promote lung maturity, even in very premature births (for instance at 24 weeks or less), such that no further treatment is required.
This Example provides exemplary protocols for producing a nanoparticle-based drug delivery system for direct pulmonary delivery of SCGB3A2. By way of example, the nanoparticles are made from gelatin, or polybutylcyanoacrylate.
Production of Gelatin-Based Nanoparticles
Briefly, in one exemplary protocol, 1.25 g of gelatin B is dissolved in 25 ml of distilled water and stirred at 600 rpm under constant heating of 40° C. Twenty-five milliliters of acetone is added to the gelatin solution. The high molecular weight gelatin is then precipitated from the solution. The supernatant containing low molecular size gelatin which is still soluble in the aqueous/organic solvent mixture is then discarded. The high molecular weight gelatin is then redissolved in 25 ml of distilled water and stirred at 600 rpm under constant heating of 40° C.; the pH of the solution is adjusted to 2.5 by adding 1N HCl; 75 ml of acetone is added to the acidic gelatin solution drop-wise, and the nanoparticles are precipitated from the solution.
One hundred and twenty-five microliters of 1 mg/ml of solution of Texas Red in acetonitrile is added and stirred for 1 hour. The particles are stabilized using 400 μl of 25% glutaraldehyde as a cross-linking agent, and the suspension is left stirring for 12 hours without heating. The remaining solvent is evaporated using a Rotavapor (for instance, IKA, Model RV 05, Staufen, Germany). The nanoparticles are purified by centrifugation at 100,000×g (for instance, in a Beckman Model J2-21) for 30 minutes, and are washed three times with distilled water. The resulting particles are re-dispersed in 25 ml of distilled water. The fluorescently-labeled nanoparticles are then stored at 4° C. and protected from light.
Production of Polybutylcyanoacrylate-Based Nanoparticles
Briefly, in one exemplary protocol, polybutylcyanoacrylate nanoparticles are prepared by an emulsion polymerization process described by Scherer et al. (1993) J. Drug Target 1, 21-27. Fifty milligrams of FITC-Dextran is added to 10 ml of 0.01N HCl. The solution is stirred at 600 rpm; 100 μl of the monomers are slowly added by pipette to the solution. The solution is then stirred for 4 hours and is protected from light; the pH is subsequently adjusted using 1N NaOH to pH 5.0. The particles are then purified from unbound dye and polymerization residuals as described for the gelatin particles, above.
Nanoparticles are then suspended in 25 ml of distilled water after centrifugation, yielding 2 mg/ml of poly-cyanoacrylate nanoparticles.
To prepare inhalable nanoparticle powders, spray-drying is a commonly practiced method (see, for instance, Bosquillon et al. (2001) J. Control. Release 70, 329-339; Mackin et al. (1997) Pharm. Sci. 3, 583-586; Vanbever et al. (1999) Pharm. Res. 16, 1735-1742). For spray-drying the nanoparticles (gelatin or polybutylcyanoacrylate), a Mini-Spray Dryer produced by Büchi Laboratoriums-Technik (Flawil, Switzerland) or the equivalent is used. The Mini-Spray Dryer operates on the principle of a nozzle spraying in a parallel-flow (the sprayed product and the drying air flow are in the same direction). The adjustable parameters include inlet and outlet temperature, solution pump flow rate, and the aspirator partial vacuum. In one exemplary protocol, the inlet air temperatures range from about 170 to 180° C., the pump flow rate is about 2 ml/minute, the aspirator is set to 40 m3/hour, and the atomizing air flow rate is about 700 l/hour (80 psi). The solution is pumped into the feeding system of the spray-dryer. The resultant powder is then blown through the cyclone separator and collected in a container. Exhaust air is extracted out of the cyclone by a vacuum pump and filtered using a fiber filter.
Five grams of SCGB3A2 are then dissolved in 75 ml of distilled water and heated up to 40° C. to increase the SCGB3A2 solubility. Then, the solution is mixed with 25 ml of either gelatin nanoparticles or polybutylcyanoacrylate nanoparticles. The glass chambers of the spray dryer are shielded from light. The powders are removed from the collector vessel and stored at room temperature under light protection.
This Example describes an exemplary method of growing lung cells on poly-DL-lactic acid scaffolds in order to engineer lung tissue for transplant (see, for instance, Lin et al. (2006) J. Biomaterials Applications 21, 109-118). Poly-DL-lactic acid (PDLLA) is a well-known super-high molecular-weight acid that has good biocompatibility and degrades following in vivo implantation (see, for instance, Lee & Gardella (2002) Analytical and Bioanalytical Chem., 373(7): 526-537). In one exemplary protocol, either dense (non-porous) 2-D films and 3-D foam scaffolds of high porosity are used. Poly-DL-lactic acid films are prepared by dissolving 0.15 g of the polymer homogeneously in 3 mL of dimethyl carbonate (DMC) at 50° C. This polymer-solvent mixture is applied to circular glass coverslips (16 mm in diameter to fit into 24-well cell culture plates) previously cleaned with acetone and alcohol. The coverslips are left at room temperature in a fume hood for 48 hours to remove the solvent and then are immersed in distilled water for 24 hours. The PDLLA films are then peeled intact from the coverslips and stuck to the base of the cell culture wells (24-well plates; for instance, Triple Red, UK) using a small amount of DMC. The PDLLA foams are prepared by thermal induced phase separation (TIPS) processing. Briefly, PDLLA with an inherent viscosity of 1.62 dL/g is used (for instance, Purasorb, Purac Biochem, Goerinchem, The Netherlands). The polymer is dissolved in DMC to give a polymer weight to solvent volume ratio of 5%. The mixture is then stirred overnight to obtain a homogeneous polymer solution. The solution is then transferred to a lypholization flask, immersed in liquid nitrogen, and maintained at −196° C. for 2 hours. The frozen solution is then transferred into an ethylene glycol bath at −10° C. and connected to a vacuum pump (10−2 Torr). The solvent is sublimed at −10° C. for 48 hours and then at 0° C. for 48 hours. The foam samples are subsequently completely dried at room temperature in a vacuum oven until reaching a constant weight, for instance, as determined using an electronic balance.
The multiwell plates containing films or foams are sterilized prior to use by exposure to UV light for 1 hour. The discs and foams are preconditioned for 9 days in basal HUES medium (Invitrogen, Paisley, UK), which includes 50% Kaighn's modification of F-12 (F-12K), 50% Dulbecco's modified Eagle's medium (DMEM; LGC, UK) with an added 10% antibiotic/antimycotic (A/A) solution (Invitrogen, UK). The medium is changed each day with a decreasing A/A concentration. Lung epithelium cells, either from primary cultures or cell lines, are seeded at a density of 2000 cells/cm2 (a total of 4000 cells per well) on to the PDLLA films in 24-well plates and grown for 1-8 days or more in the presence of 10 ng-10 mg/ml SCGB3A2, with the medium being changed each day. The PDLLA foams are seeded with lung epithelial cells and cultured for 1-8 days or more in the presence of 10 ng-10 mg/ml SCGB3A2, and the medium is changed each day. After the lung cells have grown to the desired density/maturity, the foams are washed and transplanted into an area of diseased or damaged lung tissue in a subject.
In view of the many possible embodiments to which the principles can be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as a limitation on the scope of the disclosure. Rather, the scope is in accord with the following claims. We therefore claim all that comes within the scope and spirit of these claims.